Characterization of the MIB-MIP system of different Mollicutes using an engineered Mycoplasma feriruminatoris

Characterization of the MIB-MIP system of different Mollicutes using an engineered Mycoplasma feriruminatoris | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characterization of the MIB-MIP system of different Mollicutes using an engineered Mycoplasma feriruminatoris Sergi Torres-Puig, Silvia Crespo-Pomar, Hatice Akarsu, Thatcha Yimthin, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3854399/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jun, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate virulence factor present in multiple pathogenic species of the Mollicutes , including the fast-growing species Mycoplasma feriruminatoris . The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work we analyzed the distribution and genetic relatedness between MIB-MIP systems of different Mollicutes species. Using -omics technologies, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels, are transcribed as operons controlled by four different promotors. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using oriC -based plasmids. The two proteins were functionally expressed at the surface of M. feriruminatoris , which confirms the possibility to display large functional heterologous surface proteins in M. ferirumintoris . Functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex. Biological sciences/Microbiology/Bacteriology Biological sciences/Microbiology/Microbial genetics/Bacterial genes Mycoplasma feriruminatoris genome transplantation oriC-plasmids immunoglobulin cleavage pathogenic porcine Mollicutes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bacteria of the class Mollicutes are characterized by the absence of a cell wall and numerous enzymatic pathways that were lost by reductive evolution from Gram-positive ancestors. As a result, Mollicutes have a pleomorphic cell shape and live a parasitic lifestyle to scavenge nutrients from their host. A number of Mollicutes infecting plants or animals including humans are pathogenic, such as the well-known pathogens Candidatus Phytoplasma asteris 1 , members of the “ Mycoplasma mycoides cluster” 2 and Mycoplasmoides pneumoniae and Mycoplasmoides genitalium 3 , 4 , respectively. Knowledge of their virulence traits is still scarce due to the fastidious nature of these organisms and the historical lack of genetic tools to modify their genomes. Recently, the candidate virulence factor Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system has been characterized in various Mollicutes species 5 – 7 . This system consists of at least two surface located proteins that bind and cleave the variable region of the heavy chain (Vh) of IgGs and has been shown to be active in vivo in goats infected with Mycoplasma mycoides subsp. capri ( Mmc ) 8 . Cryo-electron microscopy visualized how the two proteins bind to the Fab fragment in a “hug of death” mechanism, which is thought to interfere with antibody-antigen interactions 9 . Mycoplasma feriruminatoris ( Mferi ) is a close relative of the Mollicutes belonging to the ' M. mycoides cluster' and has been isolated from Alpine ibex and Rocky Mountain goats 10 , 11 . This species is characterized by its short doubling time compared to the slow growth typically observed in many other Mollicutes . The genome of Mferi has been recently adapted to synthetic genomics techniques 12 , including genome editing in Saccharomyces cerevisiae and genome transplantation 13 . All these features together with the simplistic nature of Mollicutes , absence of a cell wall and different genetic code have turned Mferi and other Mollicutes as a promising candidate to serve as a workhorse for industrial applications 14 such as in vivo vaccine- or drug delivery 12 , 15 , 16 . In this work we tested the pathogenicity of Mferi in domestic goats in vivo and we developed oriC -based plasmids to allow rapid introduction of genes and in cellulo expression of homologous and heterologous DNA at a high turnaround time. We also analyzed in depth the operons expressing the four MIB-MIP gene copies present in Mferi and multiple other Mollicutes species and expressed them individually to test their activity, proving that Mferi and the tools developed in this work are valuable for functional genomics of this and other Mollicutes species. Materials and Methods Strains used and culture conditions Escherichia coli Stellar cells (Clontech) or NEB 5-alpha (New England Biolabs) were used for all constructions of different oriC -plasmids and subsequent plasmid preparations. All E. coli strains were cultured in Luria Bertani (LB) medium at 37º C and shaking at 220 rpm or on LB agar plates supplemented with 100 µg mL − 1 ampicillin when necessary. Transformation of E. coli strains was achieved by using a heat-shock standard protocol. S. cerevisiae strain W303a was used to modify and propagate the Mferi genome. S. cerevisiae was cultured in Yeast Peptone Dextrose Adenine (YPDA) or Synthetic Defined (SD) broth (Formedium) depleted for tryptophan, uracil and/or histidine depending on the auxotrophic marker in use. S. cerevisiae strains were cultured at 30° C and 220 rpm. Mferi IVB14/OD_0535 14 , Mesomycoplasma hyorhinis JF5820 17 , and Mesomycoplasma hyopneumoniae Ue273 used in this study were isolated from diagnostic material at the Institute of Veterinary Bacteriology in Bern. M. hyopneumoniae Ue273 was isolated from bronchial tissue of a Swiss wild board. Mferi strains used for -omics and in vitro studies were grown at 37º C with 5% CO 2 in SP-5 medium 18 or modified Hayflick agar plates 19 supplemented with 15 µg mL − 1 tetracycline or 16 µg mL − 1 puromycin when necessary. Presence of oriC -plasmids in liquid cultures was maintained by puromycin (8 µg mL − 1 ). M. hyorhinis and M. hyopneumoniae were grown in Friis medium at 37º C. Mycoplasma capricolum subsp. capricolum ΔRE ( Mcap ΔRE) was used as a recipient for genome transplantation from yeast 20 (see ‘Genome transplantation’ section below). For the experimental infection M. capricolum subsp. capripneumoniae ( Mccp ) ILRI181 and Mferi G5847 T were grown at 37° C with 5% CO 2 in Mycoplasma Experience Liquid Medium (Mycoplasma Experience), aliquoted and stored at -80° C until used. Phylogenetic analysis The phylogenetic analysis based on the 16S rRNA gene sequences of the Mollicutes covered overall n = 7 genera, encompassing n = 27 species and n = 36 strains. The tree was built in BioNumerics v8.1 using Jukes-Cantor correction and the Neighbour Joining method. Clostridium innocuum was used as outgroup. For the phylogenetic analysis of the MIB-MIP system the translated amino acid sequences were used using the genomes described above (Supp. Table S1 ). Sequences were trimmed to a uniform size and used for analysis. Orphan copies of either the MIB or MIP were not included in the analysis. We compared the trees based on the MIB, MIP or MIB-MIP using a Pearson correlation. Unrooted phylogenetic trees were build using the model LG + F + I + G4. The trees were then plotted and manually edited in FigTree (v1.4.4). Genomic DNA extraction and Next Generation Sequencing Genomic DNA (gDNA) from Mollicutes was extracted from 20 mL cultures using the Promega Wizard Genomic DNA purification kit. The quality and quantity of the gDNA was assessed on agarose gels and using the Qubit fluorometer (Invitrogen). Subsequently, gDNA was sequenced in the PacBio sequencing platform at the Lausanne Genomic Technologies Facility at the Center for Integrative Genomics, University of Lausanne as described elsewhere (Hill et al., 2021). Briefly, DNA was sheared in a Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain 10 kb fragments and the DNA size distribution was confirmed on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). A barcoded SMRTbell library was prepared with 480 ng of gDNA using the PacBio SMRTbell Template Express Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA), according to the manufacturer's recommendations. Libraries were pooled and sequenced with v3.0/v3.0 chemistry on a PacBio Sequel instrument (Pacific Biosciences, Menlo Park, CA, USA) at 10 hours movie time, pre-extension time of 2 hours, using one SMRT cell v3. Genomes were assembled from PacBio reads using the software Flye, version 2.6 21 . Circularized genomes were polished with three rounds with the Arrow software [single-molecule real-time (SMRT) Link version 8 package]. Genomes were rotated to the first nucleotide of the start codon of the dnaA gene, and annotated using Prokka, version 1.13 22 . M. hyopneumoniae Ue273, Mesomycoplasma ovipneumoniae 14KM848 and Mferi ΔMIB-MIP sequences are deposited as BioProject PRJNA1062711. Transcriptomic analysis and primer extension Mferi for transcriptomics analysis was carried out as previously described 23 . Briefly, RNA from three biological replicates of Mferi was extracted from liquid culture using the Zymo Research Quick-RNA Fungal/Bacterial Miniprep kit. RNA quality was assessed at the Lausanne sequencing platform on a Fragment Analyzer (Agilent Technologies). Libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina), excluding the polyA selection step and using a unique dual indexing strategy. Ribosomal rRNA depletion was carried out with QIAseq FastSelect–5S/16S/23S kit (Qiagen). Libraries were quantified by QubIT, Life Technologies and their quality was assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 1.92 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 2 x 150 cycles (paired end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (v. 2.20, Illumina). For primer extension analyses, total RNA was extracted from 10mL culture at early stationary phase using the RNAqueous Total RNA Isolation kit (ThermoFisher Scientific) following manufacturer's instructions. Primer extension reactions were performed using 20–25µg of total RNA, the SuperScript IV First Strand Synthesis system (ThermoFisher Scientific) and 6-Carboxyfluorescein (6-FAM) labeled primers (Supp. Table S2), as previously described 24 , 25 with few modifications. Briefly, 20–25µg of total RNA were mixed with 0.5µL of 5µM 6-FAM labelled primer and 1.5µL of dNTPs in a 20µL final volume reaction. This mix was incubated for 5 min at 65ºC in a thermocycler and subsequently cooled down on ice. In a separate tube, a 10µL reaction mix was prepared by adding 6µL 5x SuperScript IV RT buffer, 1.5µL 0.1M DTT, 60U of RNase Inhibitor, and 1µL of SuperScript IV Reverse Transcriptase. This reaction mix was added to the tube containing the RNA and primer on ice and mixed by gently pipetting before incubating at 55ºC for 30 min in a thermocycler. The reaction was inactivated at 80ºC for 10 min prior addition of 1µL RNase H and incubation at 37°C for 1h. cDNA was precipitated with 0.1 volumes of 3M sodium acetate and 2.5 volumes of absolute ethanol. The cDNA pellet was washed in 70% ethanol, resuspended in 10µL Hi-Di formamide (ThermoFisher Scientific) and kept protected from light exposure at room temperature. Samples were separated and analyzed in an ABI3730XL instrument at Microsynth AG (Switzerland) using ROX size standards. Proteomics analysis Proteomics analyses were performed as previously described 23 . Briefly, the same three biological replicates of mycoplasmas used for transcriptomics were harvested by centrifugation at 4,000 x g at 4°C for 15min. Pellets were washed three times in ice-cold PBS and stored at -80°C until further use. The cells pellets were thawed on ice and subsequently lysed via resuspension in 8M urea/ 100mM Tris-HCl and precipitated overnight at -20˚C in acetone. Protein pellets were air-dried at room temperature for 15 min before being reconstituted in 8M urea/50mM Tris-HCl. A protein aliquot corresponding to 10µg protein was trypsinized overnight at room temperature in digestion buffer and subsequently stopped by adding 1% (v/v) tri-fluoroacetic (TFA). Three repetitive injections of aliquots corresponding to 500ng of trypsinized proteins were processed by liquid chromatography (LC)-MS/MS (PROXEON coupled to a QExactive HF mass spectrometer, ThermoFisher Scientific). Mass spectrometry-derived proteomic data were analyzed against custom-made databases by Transproteomic pipeline (TPP) tools 26 . The four database search engines Comet 27 , Xtandem 28 , MSGF 29 and MyriMatch 30 were used and each search was followed by the application of the PeptideProphet tool 31 . The iProphet 32 software was subsequently used to summarize the search results, which were filtered at the false discovery rate of 0.01. Protein identifications were exclusively accepted if at least two of the search engines agreed on the identification. The decoy approach was used for custom databases containing standard entries and protein inference was investigated using ProteinProphet. For those protein groups accepted by a false discovery rate filter of 0.01, a Normalized Spectral Abundance Factor (NSAF) 33 was calculated based on the peptide to spectrum match count. Shared peptides were considered by a method published elsewhere 34 . Plasmid construction All plasmids used in this study were constructed using the NEBuilder HiFi DNA Assembly kit (New England Biolabs), following manufacturer’s instructions. The plasmid pIVB03 was constructed by replacing the origin of replication of Mmc GM12 present in pMYCO1 35 by the origin of replication of Mferi type strain G5847 T 10 , amplified with primers #001 and #002 (Supp. Table S2). The plasmid pIVB04 is a derivate of pIVB3 in which the tetM marker under the control of the spiralin promotor (pS’) was replaced by pS’ pac marker, amplified from the gDNA of Mcap ΔRE strain 20 using primers #003 and #004. The plasmid pIVB06 is a derivate of pIVB04 in which the orientation of the pS’ pac marker was switched. This switch was performed by amplifying the pIVB04 backbone without the pS’ pac marker using primers #007 and #008 and the pS’ pac marker using primers #005 and #006. Plasmid pIVB08 is a derivate of plasmid pIVB03 and was constructed by assembling the pS’ tetM marker amplified with primers #011 and #012 with the pIVB03 backbone amplified in two parts using primers #009 with #010, and #013 with #014. The plasmid pIVB09 was built by replacing the pS’ tetM cassette from pIVB08 by the pS’ pac marker, employing primers #009 and #016 for the amplification of the pIVB08 backbone and primers #011 and #015 to amplify the pS’ pac marker. All plasmids carrying MIB-MIP copies of the different Mollicutes have the pIVB09 as a backbone, amplified using primers #017 and #018. The MIB-MIP gene pairs and their natural promoter regions were amplified from gDNA of each respective host strain ( Mferi IVB14/OD_0535, M. hyopneumoniae Ue273, M. hyorhinis JF5820) using primers listed in Supp. Table S2. To generate plasmids expressing MIB-MIP gene copies with the promoter region of the first MIB-MIP gene copy of Mferi IVB14/OD_0535 ( P MM1mfe ), the pIVB09 backbone containing the P MM1mfe was amplified using primers #017 and #051 and each MIB-MIP copy with their respective primer set listed in Supp. Table S2. Plasmid carrying the tagged MIB-MIP copy number 4 from Mmc was created amplifying the promoter sequence of the first MIB-MIP copy of Mmc using primers #057 and #058, and each gene with primers #059 and #060, and #061 and #062. Plasmids bearing codon-optimized MIB-MIP gene pairs tagged with 6xHis and FLAG, respectively, were created by cloning each gene copy, which was custom synthesized (GenScript). Optimization was carried out by using the Optimizer tool 36 . All plasmids generated in this study are listed in Supp. Table S3 and the proper manipulations were sequence verified by Sanger sequencing at Microsynth AG (Switzerland). CReasPy-Cloning of M. feriruminatoris genome A mutant strain of Mferi IVB14/OD_0535 without the four MIB-MIB gene pairs (ΔMIB-MIP) was generated using the CReasPy-Cloning method 37 . Briefly, two different gRNA sequences were designed by using the “CRISPR Guides” tool available in the Benchling work environment ( https://benchling.com ). Target sequences with the highest on-target score and the lowest off-target score were selected. Two gRNAs were designed to target the genes coding for the 4 copies of the MIB-MIP system in Mferi (locus_tags MF5583_00301 - MF5583_00308), by using complementary primers (#084–087). The corresponding pgRNA plasmids (pgRNA-1MIBMIP, pgRNA-2MIBMIP) were constructed following the protocol described elsewhere 38 . Plasmids were sequence verified using Sanger sequencing at Microsynth AG (Switzerland) and purified using QIAprep Miniprep Spin Kit (Qiagen). Plasmids were then transformed into S. cerevisiae W303a-eSpCas9 via lithium acetate transformation 39 and transformants were selected on SD-Trp-Ura medium (Takara). Recombination templates containing the yeast elements (YCp1.1) and the tetracycline resistance cassette (pS' tetM ) were produced by PCR amplification of the ARS4/CEN6/HIS/pS’ tetM loci from the plasmid pMT85–PSTetM-ARSCenHis-pRS313 18 using primers #082 and #083 with the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and purified using the High Pure PCR Product Purification Kit (Roche). The resulting recombination template contained sequences with 50bp homology to the regions flanking the MIB-MIP locus of the Mferi genome. All primers are listed in Supp. Table S2. Mferi genomes were isolated and introduced in S. cerevisiae W303a-eSpCas9 pgRNA-1MIBMIP or pgRNA-2MIBMIP by spheroplast transformation 40 , as previously described 12 . Genome transplantation into Mcap ΔRE recipient cell The genome IVB14/OD_0535::YCp-ΔMIB-MIP maintained in S. cerevisiae as a Yeast Artificial Chromosome (YAC) was transplanted into Mcap ΔRE strain as previously described 12 , 20 . Briefly, Mcap ΔRE was grown in SOB + medium until early stationary phase (pH 6.5), washed in 10 mM Tris 250 mM NaCl pH 6.5 and resuspended in cold 0.1 M CaCl 2 . Transplantation was carried out mixing SP-5 without serum and agarose plugs containing the modified genome with the resuspended cells in 2X Fusion Buffer (20 mM Tris, 20 mM MgCl 2 , 500 mM NaCl, 10% PEG 6000 pH 6.5). Mixtures were incubated statically for 90 min at 30°C and then plated in SP-5 plates supplemented with 15 µg mL − 1 tetracycline. The resulting transplanted mutant strains were subjected to multiplex and simplex PCRs to confirm integrity of the genome (see primer list in Table S2). Additionally, the genomes of mutant strains were verified via Illumina-type next generation sequencing and mapping assembly to the Mferi IVB14/OD_0535 parental strain 14 . Growth rate determination Growth rate of the different mycoplasma strains was assessed by color-changing units (CCU) per mL as well as colony-forming units (CFU) for up to 20 h every 60 min. Briefly, 100 mL SP-5 at pH = 7.5 containing 10 2 cells mL − 1 were incubated at 37º C and 5% CO 2 and small aliquots were removed every hour. Each aliquot was serially diluted and distributed in 200 µL volumes using 96-well plates (for CCU calculation) and plated as spot dilutions in SP-5 agar plates (for CFU calculation). Color change and colonies were assessed after 2 days of incubation. To better compare the growth rate between wild-type (WT) and mutant strain, no additional antibiotics were added to the SP-5 medium during the experiment. Growth curves were plotted, and the growth rates were calculated using GraphPad Prism v9.0.0 Transformation of M. feriruminatoris and screening of mutants The transformation protocol used to transform oriC -plasmids into Mferi IVB14/OD_0535 was adapted from the one used for transformation of Mmc 20 , with some modifications. Mferi was grown overnight (O/N) in SP-5 with pH adjusted to 8.0 (SP-5 pH8 ) until late logarithmic phase (~ pH = 7.0) to achieve the highest total CFU mL − 1 (4 mL culture/ transformation). Cells were cooled on ice, pelleted at 4,200 x g for 15 min at 4º C and washed once in Sucrose/Tris Buffer (0.25 M Sucrose, 10 mM Tris-HCl, pH = 7.0). Each cell pellet was resuspended in 400 µL cold CaCl 2 0.1 M and kept on ice for 30 min. In a 50 mL falcon tube, 10 µL of plasmid (600–1000 ng µL − 1 ) were added to 400 µL Fusion Buffer 2X (0.5 M Sucrose, 20 mM Tris-HCl, 40% PEG8000, pH = 7.0) and left at room temperature. After an incubation of 30 min, the 400 µL cell mix was added into the Falcon tube containing the Fusion Buffer 2X as well as the plasmid and mixed gently. The reaction was left incubating at 30º C for at least 25–30 min. Then, fusion was stopped by adding 9 mL of cold SP-5 and inverting the tube once. Cells were recovered by centrifugation at 4,200 x g for 15 min at 10º C and the supernatant was carefully discarded. The cell pellet was resuspended in 1 mL fresh SP-5 pH8 and incubated at 37º C for 45–60 min before plating on selective agar plates. Colonies of transformants were visible after 48 h but were picked up on day 3–4 after transformation in 1.5 mL microcentrifuge tubes containing 1 mL SP-5 pH8 with the appropriate antibiotic (i.e 16 µg mL − 1 puromycin or 15 µg mL − 1 tetracycline). Transformants were passaged three times before screening. Proper transformants were verified by PCR of cell lysates obtained as previously described 18 , using the oligonucleotides listed in Supp. Table S2. MIB-MIP-derived IgG cleavage analysis IgG cleavage assays were performed as described in 5 , 9 , with some modifications. For all Mferi strains, cultures were grown O/N in SP-5 pH8 with antibiotic selection, if necessary, until stationary phase. Next day, 1mL fresh SP-5 pH8 medium was inoculated with 50 µL of the O/N cultures maintaining the antibiotic selection and grown at 37ºC and 5%CO 2 until late logarithmic phase (~ 10 9 cells). Cells were spun down at 7,000 x g for 10min, washed once with SP-5 w/o serum 18 and spun down again in the same parameters. Pellets were resuspended in 35µL of SP-5 w/o serum containing 250 ng µL − 1 of purified IgG from goat or swine serum (Sigma-Aldrich) and incubated at 37º C for 45 min. Then, cells were pelleted at 7,000 x g for 10min, and supernatants were recovered and mixed with 7µL 6x Laemmli buffer before boiling at 100º C for 10 min. Supernatants were separated in a 10% SDS-PAGE and transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad). Membrane was blocked in PBS with 5% skimmed milk (Becton Dickinson) and 0.05% Tween-20 (Sigma). Antibodies and working dilutions are listed in Supp. Table S4. Membrane was developed using SuperSignal West Pico PLUS Chemiluminiscent substrate (ThermoFisher Scientific), following manufacturer’s instructions. In the case of M. hyorhinis and M. hyopneumoniae , IgG cleavage determination was performed similarly, but cells were grown in Friis until early stationary phase and no subculture step was performed. Besides, incubation with IgG was extended to 2 h ( M. hyorhinis ) or 3 h ( M. hyopneumoniae ). Analysis of expression of MIB and MIP Expression of C-terminal tagged MIB and MIP was analyzed by immunoblot using anti-6xHis and anti-FLAG antibodies (Table S4). Bacterial cells were washed twice in sterile PBS before suspended in 100 µL. Lysates were obtained by adding 20 µL 6x Laemmli buffer and boiling at 100° C for 10 min. Lysates were separated in a 7.5%-8% SDS-PAGE before blotting. The immunoblots were performed as described above. Statistical analysis All analyses were carried out using GraphPad Prism (v9.0.0). One-way ANOVA tests and Tukey’s comparative tests were performed to assess significance and calculate p values when indicated. In vivo infection of domestic goats The experimental infection of goats was performed in compliance with the Swiss animal protection law (TSchG SR 455; TSchV SR 455.1; TVV SR 455.163) under the cantonal license BE67/19. The experiments were reviewed by the cantonal committee on animal experiments of the canton of Bern, (Switzerland) and approved by the cantonal veterinary authority (Amt für Landwirtschaft und Natur LANAT, Veterinärdienst VeD, Bern, Switzerland). The infection trial was carried out at the Institute of Virology and Immunology (Mittelhäusern, Switzerland) using six female goats in total, aged between 1 and 4 years old and weighting 50–60 kg and randomly split into two groups of three animals. Animals were infected intranasally with 1 mL 10 8 color changing units mL − 1 (CCU mL − 1 ) of Mferi strain G5847 T , or Mccp strain ILRI181 on two consecutive days as described recently 41 . One mL of mycoplasma culture was atomized using a 1 mL syringe attached with a MAD Nasal™ Intranasal Mucosal Atomization Device (Teleflex, UK) and 500 µL of aerosol was applied to each nostril. At 4 days post-infection (dpi), each animal was infected transtracheally with the same number of color changing units (10 8 CCU mL − 1 ). One mL of culture was followed by flushing with 5 mL sterile phosphate buffered saline. For the duration of the experiment, the animals were housed in a high containment facility, one stable per group of infection. The animals were kept on straw with a local ambient temperature of 20–22˚ C. Before and after Mferi or Mccp infection, body temperature and clinical status was monitored daily. The clinical status was assessed by a veterinarian, always the same person to ensure unbiased clinical assessment. The rectal body temperature was measured with a digital thermometer, whereas respiratory and heart frequencies employed a stethoscope. Differential blood cell counts were determined from EDTA blood samples using an automated hematology analyzer (VetScan HM5, Abaxis). Animals were euthanized when endpoint criteria were reached or at the end of the trial and subjected to postmortem analysis. Results Development of replicative plasmids for M. feriruminatoris The genetic tools currently available for manipulation of Mferi are limited and only the introduction and modification of the bacterial genome in yeast has been reported, which is technically challenging and time consuming. To accelerate the shuttle in of genes into Mferi , we decided to generate replicative plasmids based on the origin of replication ( oriC ) sequence of the chromosome (Fig. 1 A), as previously reported for other Mollicutes species 35 , 42 – 44 . We adapted the oriC-plasmid pMYCO1 for use in Mferi by exchanging the oriC region of Mmc by the oriC region of Mferi strain G5847. This first oriC -plasmid, named pIVB03, could be successfully transformed in Mferi and promoted resistance to tetracycline at 15 µg mL − 1 . However, as Mferi strains engineered in yeast already have a tetracycline resistance cassette, we developed a derivative plasmid of pIVB03 named pIVB04 carrying the puromycin acetyl transferase ( pac ) gene, which was reported to confer resistance to puromycin in other closely related Mollicutes species 45 . This puromycin resistance-conferring plasmid was transformed but did not produce puromycin-resistant colonies carrying pIVB04 (Fig. 1 B). This negative outcome was changed by switching the direction of the pac gene (Fig. 1 A), which became plasmid pIVB06, and allowed the recovery of resistant colonies at 16 µg mL − 1 puromycin. In other Mycoplasma species, oriC -plasmids have been shown to be more stable when reducing the length of the oriC region or by deleting the dnaA gene 19 , 46 . Therefore, we modified the oriC -plasmids pIVB03 and pIVB06 by replacing the dnaA gene of Mferi by the antibiotic resistance cassette, generating streamlined oriC-plasmids with resistance to tetracycline (pIVB08) or to puromycin (pIVB09) (Fig. 1 A). These plasmid versions transformed with an efficiency up to four times higher than their parental plasmids with dnaA counterparts (Fig. 1 B). They can be used for subsequent mutant phenotype complementation studies and as versatile backbones for heterologous expression systems reported in this study. MIB-MIP tandem gene copies are widespread, divergent, and horizontally transferred among the Mollicutes The MIB-MIP system is widespread between different members of the Mollicutes 5 , but little is known about their diversity. We aimed at studying the occurrence and diversity of the different existing MIB-MIP cleavage systems in different phylogenetic clades of the Mollicutes . Therefore, we selected Mollicutes genomes including the ones reported in this study and analyzed the presence of the MIB-MIP system. Altogether, we included a total of 34 genomes from 23 different species (Fig. 2 A). We analyzed the sequences of a total of 70 genes coding for putative MIB proteins and 71 genes encoding putative MIPs. In most genomes analyzed, both MIB and MIP counterparts were found adjacent in the same genetic locus, most likely forming a single transcriptomic unit. Occasionally we observed “orphan” MIB-encoding genes without a MIP-encoding gene in its vicinity. However, in those genomes with orphan MIBs, there was always another MIB-MIP pair present elsewhere in the genome, suggesting that Ig cleavage activity can potentially still occur. To facilitate the analysis, only the paired MIB-MIP proteins were included in the construction of the phylogenetic trees and the analyses (Fig. 2 A and Figure S1 , Supp. Table S1 ). An initial scrutiny of the phylogenetic relations between MIB-MIP proteins of different species revealed three major distinctive branches, (i) encompassing Mycoplasma and Mycoplasmopsis species infecting ruminants; (ii) Ureaplasma , Metamycoplasma and some MIB-MIP pairs of porcine Mesomycoplasma species; and (iii) a more distant branch containing the MIB-MIP pairs of Mycoplasmoides and Mesomycoplasma species. Interestingly, we detected several examples of closely related MIB-MIP systems between Mollicutes species that are phylogenetically very distant but share the same hosts, such as Ureaplasma species and Metamycoplasma hominis , Mycoplasmoides gallisepticum and Mycoplasmopsis synoviae , or Mycoplasmopsis pulmonis and Metamycoplasma arthritidis , infecting humans, poultry, or rodents, respectively (Fig. 2 A and 2 B). This data strongly suggests that these genes have been acquired through horizontal gene transfer between distantly related Mollicutes infecting the same host. Species belonging to the ‘ M. mycoides cluster’ and their close relatives have multiple pairs located adjacent to each other in the same chromosomal locus. Here we show that there is a strong conservation between each pair in the same position, suggesting that multiple copies of this tandem system were present in a common ancestor of these mycoplasmas. Furthermore, there is a significant difference between the two strains of Mferi analyzed, as the MIB-MIP pairs of the type-strain G5847 T are similar to the ones present in Mccp , while the MIB-MIP pairs of the IVB14/OD_0535 strain cluster closely to the ones of the other members of the ‘ M. mycoides cluster’. Remarkably, the two paired MIB-MIP copies of M. hyopneumoniae , located on different chromosomal loci, are highly divergent, with one MIB-MIP being more similar to the only system present in M. hyorhinis and the other more similar to MIB-MIP present in Mesomycoplasma ovipneumoniae (Fig. 2 A). Overall, our analysis shows how widespread these tandem systems are within the Mollicutes , with high sequence divergence and multiple possible gene acquisitions, particularly between species sharing the same hosts. Analysis of MIB-MIP expression in M. feriruminatoris We studied the expression of each MIB-MIP gene pair of this species by transcriptomics and proteomics analyses to see whether the gene pairs are organized as operons and to identify different promotors that can be used to express MIB-MIPs from other Mollicutes species. Exploration of the transcriptomic data showed that each MIB-MIP pair constitutes an individual transcriptional unit, with the first and last pairs (MM1 mfe and MM4 mfe ) being expressed at higher levels than the other two pairs (MM2 mfe and MM3 mfe ) (Fig. 3 A). Moreover, all MIB-MIP gene pairs are among the mid-to-high expressed genes of Mferi , with no apparent differences between MIB and MIP expression (Fig. 3 B). However, when analyzing the proteomics data, MIB-MIP pairs are not among the highly expressed proteins of the cell highlighting the lack of correlation between RNA and protein levels (Fig. 3 C). Besides, the expression levels of all MIP copies of each pair are significantly higher expressed compared to their MIB counterparts. Given the similar disposition and number of the different MIB-MIP gene pairs between Mferi IVB14/OD_0535 and Mmc GM12, we also analyzed transcriptomics and proteomics data of GM12 obtained in a previous study 23 . Our results showed a similar mRNA expression trend for each MIB-MIP pair, with the first tandem of genes being expressed higher than the rest (Supp. Fig. S2). Moreover, proteomics data showed that most MIP proteins are expressed at higher levels than their respective MIB counterparts, with most of them being not reliably detected (Supp. Fig. S2). Overall, these results suggest that each MIB-MIP system constitutes an individual expression unit and that expression of the different tandem of genes is species-specific, despite having similar genetic organization or distribution in the different chromosomes. Identification of MIB-MIP promoter and terminator regions To further characterize the transcriptional profile shown by the RNAseq analysis of the MIB-MIP locus of Mferi , we decided to experimentally determine the transcriptional start sites (TSSs) present in that genomic area by primer extension. We could only reliably detect the TSS of the last MIB-MIP gene tandem encoded in Mferi , while no clear signal could be seen for the other gene pairs (Fig. 4 A). The determination of the TSS of the elongation factor Tu gene ( tufB ) was used to establish the experimental conditions. Moreover, genetic analysis of the downstream regions of MIB-MIP gene clusters in a number of members of the ' Mycoplasma mycoides cluster' revealed the presence of inverted repeat elements with a structure reminiscent of rho-independent terminators (Fig. 4 B). These elements are 32-34bp long and are located very close to the stop codon of the MIP genes. The last MIB-MIP gene pair is always devoid of this downstream element in all studied species, suggesting that it is part of a larger operon unit that also comprises putative ATPase-coding genes located downstream of the MIB-MIP cluster. The consensus sequence of these elements was determined as 5’TA(A/C)NATCCTTT(A/G)G-NT(A/T 2 )T(A/T 2 )-CTAAAGGATTTTT using all available sequences (Fig. 4 C). Employing RNAfold 47 , the RNA structure of these downstream elements was predicted to be a small hairpin with a minimum free energy of approximately − 12.70 kcal mol − 1 (Fig. 4 C). Generation and characterization of a M. feriruminatoris ΔMIB-MIP strain To examine the effectiveness and versatility of the new oriC -plasmids developed for Mferi we decided to test expression of the recently identified MIB-MIP cleavage systems. Mferi strain IVB14/OD_0535 has several genes coding for a total of four MIB-MIP tandem systems clustered in a single chromosomal locus 14 , in a similar disposition as in Mmc GM12 5 (Fig. 5 A). To assess activity of each unique MIB-MIP gene pair of Mferi using oriC-plasmids in cellulo , we aimed at the generation of a ΔMIB-MIP knock-out mutant by cloning and modifying the genome of Mferi in yeast prior transplantation of the modified genome to a new mycoplasma cell. The genome of Mferi IVB14/OD_0535 was transformed into S. cerevisiae carrying the necessary plasmids to replace the chromosomal locus coding for the four MIB-MIP gene pairs (MF5583_00301 to MF5583_00308, ~ 20Kb) by a recombination template using the CReasPy-Cloning method 37 . YACs containing the modified genome were transplanted into an Mcap ΔRE recipient cell to obtain the desired Mferi ΔMIB-MIP strain (Figure S3). This knock-out mutant could grow similarly to the WT strain in the absence of antibiotics (Fig. 5 B), with a doubling time of 50 ± 2min compared to 45 ± 3min of the WT in SP-5 (Fig. 5 C). Besides, the ΔMIB-MIP phenotype was confirmed by an IgG cleavage assay in which this mutant lost the ability to cleave the heavy chain of purified goat IgGs (Fig. 5 D, lane 4), compared to the wild-type strain (Fig. 5 D, lane 3). Expression of native MIB-MIP gene pairs of M. feriruminatoris in trans Each unique MIB-MIP gene pair of Mferi was cloned in a pIVB09 backbone under the control of their own natural promoters previously determined by RNAseq or primer extension. The newly constructed Mferi ΔMIB-MIP strain was transformed with each of these plasmids individually. Positive clones were exposed to goat IgGs to assess cleavage activity (Fig. 5 D). Despite analyzing multiple clones harboring each MIB-MIP gene pair (data not shown), we could only detect IgG heavy chain cleavage in clones expressing the first and last gene tandems of the cluster (MM1 mfe and MM4 mfe ), while other clones expressing the other MIB-MIP pairs (MM2 mfe and MM3 mfe ) showed marginal cleavage or activity below the detection limit. As the transcriptomics data suggested that the MM1 mfe and MM4 mfe gene tandems were expressed at higher levels than MM2 mfe and MM3 mfe , and that expression from the promoter region of MM1 mfe (P MM1mfe ) had no apparent interplay with any rho-independent terminator or upstream regulatory sequences, we decided to reintroduce the MM2 mfe and MM3 mfe gene pairs under the control of P MM1mfe in a ΔMIB-MIP genetic background. Under these conditions, all MIB-MIP gene pairs could be expressed, and mutants showed clear IgG cleavage activity (Fig. 5 E), suggesting that all MIB-MIP gene pairs are functional in cellulo , and that the P MM1mfe region was sufficient to drive expression of two relatively large membrane-associated proteins organized in an operon. In vivo IgG cleavage by M. hyopneumoniae and M. hyorhinis Cleavage of immunoglobulins by the MIB-MIP system has been reported in Mollicutes of the formerly known ' Spiroplasma phylogenetic group', i.e Mmc 9 or Mferi 12 , but never in Mollicutes species like Mesomycoplasma spp. To determine if important porcine pathogens such as M. hyopneumoniae or M. hyorhinis can target and cleave host IgGs, we analyzed immunoglobulin cleavage activity of two strains isolated in Switzerland. Genome analysis revealed that M. hyopneumoniae Ue273 contains two complete MIB-MIP gene pairs and a single orphan MIB gene, while M. hyorhinis JF5820 only contains a single MIB-MIP gene pair 17 (Fig. 6 A). This contrasts with many Mollicutes species of the ‘Spiroplasma phylogenetic group’, where all MIB-MIP gene copies are clustered in a single chromosomal locus containing 3–4 complete MIB-MIP gene pairs. Interestingly, neither of the different MIB-MIP copies in M. hyopneumoniae were downstream followed by an ATPase gene cluster, as it is the case in most Mollicutes species. This ATPase gene cluster in M. hyopneumoniae is found in a different chromosomal location instead, seemingly controlled by a DNA slippage mechanism in a similar fashion as antigenic or phase variation switches present in Mollicutes (Figure S4) (Citti 2010). Incubation with purified commercial IgGs isolated from naïve pig serum showed cleavage activity by both pathogens M. hyorhinis and M. hyopneumoniae (Fig. 6 B), with the heavy chain of the immunoglobulins being targeted. Heterologous expression of MIB-MIP gene pairs of other Mollicutes The availability of a ΔMIB-MIP strain together with a vector capable of expression of MIB-MIP pairs in trans in Mferi prompted us to complement this strain with MIB-MIP systems from other Mollicutes species. First, we cloned the last gene tandem of the MIB-MIP operon of Mmc GM12 (MM4 Mmc ) under the control of the promoter region of the first gene tandem of the same species (P MM1Mmc ), mimicking a similar disposition performed in situ at the chromosomal location in a previous work 9 . This construction was transformed in the ΔMIB-MIP strain and positive clones exhibit restored capacity to cleave goat IgGs (Fig. 6 C, lane 6). Hereafter, we cloned the two complete MIB-MIP gene pairs and the single MIB-MIP gene tandem from M. hyopneumoniae Ue273 (MM1 Mhp and MM2 Mhp ) and M. hyorhinis JF5820 (MM Mhr ) in a pIVB09 backbone. In a first attempt, we introduced each MIB-MIP set under the control of their natural promoters, as previously done with the MIB-MIP genes of Mferi and Mmc . However, despite obtaining similar number of transformants carrying the different oriC -plasmids, no IgG cleavage was detected (Fig. 6 C). To facilitate recombinant expression, we adapted all the MIB-MIP coding sequences to the codon usage of Mmc GM12, the closest species of the ‘ Mycoplasma mycoides cluster’ to Mferi with an available characterized codon usage table (kazusa.or.jp), and replaced the natural promoters with the P MM1Mfe , which proved capable of generating mRNA of similar length as previously shown in this study. However, transformants carrying these new constructs could neither cleave goat nor porcine IgGs (Fig. 6 C), suggesting that the system was not active or could not be correctly exported, folded or displayed at the membrane of Mferi cells. To further investigate this, we decided to clone in pIVB09 tagged-versions of the MIB-MIP systems of Mferi (MM4 Mfe ), Mmc (MM4 Mmc ) and the single MIB-MIP system of M. hyorhinis (MM Mhr ) to track protein expression by immunoblotting. All MIB genes were fused with a C-terminal 6xHis tag, while their MIP counterparts were tagged with a C-terminal FLAG tag. Analysis of Mferi ΔMIB-MIP strains carrying these plasmids showed that neither of the proteins forming the MIB-MIB system of M. hyrorhinis was expressed in these conditions (Fig. 7 A), which explained the lack of IgG cleavage showed previously. Sequence analysis of the MIB-MIP systems of Mferi , Mmc and M. hyorhinis showed significant differences in the N-terminal residues (Supp. Fig. S5), which could prevent export of these proteins to the cell surface. To test this, we replaced the predicted signal peptides of the tagged MIB-MIP system of M. hyorhinis with the ones present in the MIB-MIP pair 1 of Mferi . Strains carrying this construction could correctly express the protease component of the MM Mhr , but not the binding protein (Fig. 7 B). Goats infected intranasally and transtracheally with Mycoplasma feriruminatoris did not develop disease Mferi is considered a promising candidate for the development of a vaccine chassis 12 . However, Mferi has only been isolated from wild caprinae 10 , data regarding its pathogenic potential in closely related domestic animals are absent. Therefore, we decided to assess the pathogenicity of the type-strain G5847 T of Mferi as the representative member of the species. We used a challenge model established for the phylogenetically related species Mmc 8 and modified for Mccp 41 , which is robust and reproducible 48 . Positive control was the highly virulent Mccp ILRI181 49 . Clinical evaluation was assessed daily and was carried out 10 days pre-infection up to 25 days post-infection (dpi). Goats infected with Mferi showed no clinical signs in contrast to animals infected with Mccp , which showed onset of clinical disease including elevated body temperature at 6–8 dpi (Fig. 8 ). This was followed by high fever (> 40.5°C for all animals), associated with respiratory distress, coughing and wheezing (8–10 dpi), less movement and reduced intake of food. All criteria considered, this clinical evaluation led to a severity grade of 3 at 10 dpi; consequently, the three animals infected with Mccp were euthanized (Supp. Fig S6). During the course of infection, we monitored the hematological parameters. All animals infected with either species did not show a clear difference in the hematological parameters compared to their baseline levels prior infection (Supp. Fig S6). Postmortem analysis did reveal CCPP-typical pathomorphological changes including the detection of Mccp , while the animals infected with Mferi did not have any lesions pointing towards Mferi -related disease and Mferi could not be isolated from the animals. Discussion Mferi has so far only been isolated from wild ruminants such as Alpine ibex 10 , 11 . The ability to modify its genome using synthetic genomics tools 12 , the absence of the cell wall, its glycosylation capacity and its favorable growth attributes that compare to model organisms such as E. coli make it an appealing candidate for vaccine- and drug delivery especially with reference to the respiratory tract or cancer treatment 50 . In this work, we investigated the pathogenicity of Mferi in domestic goats using an infection model that has been successfully used for phylogenetically closely related mycoplasmas. Mollicutes are reported to have high species tropism this should be confirmed for Mferi in an in vivo experiment. Our data do not point to pathogenicity in domestic goats and therefore this organism is unlikely to infect even phylogenetically more distant organisms, which is important for safety concerns. Only one infection route was tested, which is the main one in closely related bacteria of the ‘ M. mycoides cluster’ 51 . Goats challenged with Mccp , as expected, reached endpoint criteria at 10 dpi and were euthanized. Mferi could not be isolated from animals challenged with the latter, while Mccp was isolated from pathomorphological lesion of the animals infected with it. The absence of Mferi from the post-mortem tissues investigated supports the fact that the animals cleared Mferi from the system. Surface expression of heterologous antigens in Mferi is desirable for future antigen presentation in a live vaccine chassis. We aimed to investigate heterologous surface expression and focused our work on the MIB-MIP system. To get an idea of the sequence conservation of the MIB-MIP system in Mollicutes we analyzed the presence and genetic identity in different species. It has been shown that genes homologous to the MIB-MIP system of Mmc were present frequently as several repeated gene copies in most species of Mycoplasma , Mycoplasmopsis , Metamycoplasma and Mesomycoplasma , and a token presence in Mycoplasmoides spp. 5 . Here, we show that these systems are highly divergent in the different Mollicutes , with several copies being potentially exchanged between distant species sharing the same host. Horizontal transfer in these bacteria has been mainly attributed to the presence and activity of integrative-conjugative elements (ICE), first described in Mycoplasmopsis fermentans 52 and later studied in depth in Mycoplasmopsis agalactiae 53 – 55 and Mycoplasmopsis bovis 56 . Some species of Mollicutes carry MIB-MIP tandem genes in close vicinity of ICE sequences, like the case of Mmc GM12 8 , which could explain the dissemination of these immunoglobulin cleavage systems between species inhabiting the same niche. No traces of vestigial ICE could be found near the MIB-MIP sequences of M. synoviae , M. pulmonis , M. arthritidis or M. gallisepticum , however other ICE-independent conjugation mechanisms seem to exist among the Mollicutes 57 , 58 and could be used to transfer specific genomic sequences between different species. Another possibility could be the transfer of this genetic material via phage infection, as some species like M. pulmonis or M. arthritidis are susceptible to phage attacks 59 , 60 , although genetic exchange via viral transduction has never been reported in Mollicutes to the best of our knowledge. Next, we developed transformation protocols and oriC-type plasmid vectors to shuttle antigen-encoding genes into Mferi and to accelerate the testing of heterologous protein expression. Our growth curves of the wild-type strain of Mferi showed the rapid decline of viable cells after 20h of cultivation in SP-5 medium, coinciding with medium acidification below pH7. This characteristic contrasts slightly Mmc , a closely related relatively fast-growing Mycoplasma species, which can survive and maintain high bacterial titers for longer time in acidic conditions. Loss of viability upon acidification in mycoplasmas is not exceptional 61 , thus the differences in low pH tolerance between Mferi and Mmc could be attributed to distinctive metabolic capabilities 10 or growth requirements in both species. This reduced tolerance towards lower pH prompted us to adapt the standard transformation protocols for mycoplasmas of the ' M. mycoides cluster', which use cells harvested at pH 6.2–6.5 and PEG solutions buffered at a similar pH, for transformation of Mferi . Most transformable Mollicutes species have higher transformation efficiencies when harvested at late-log phase 62 . By increasing the initial pH of the SP-5 medium from 7.5 to 8, we could significantly increase the bacterial titers of Mferi after an overnight growth to 1-3x10 9 CFU/mL at pH 7, right before cell titer decline, optimizing transformation efficiencies for this bacterium. Thus, we also adapted the pH of all transformation solutions to pH 7 to mimic the medium conditions at the harvesting point. In this work we developed a series of replicative plasmids based on the modification of the origin of replication of the type-strain G5847 T that can be easily used in Mferi . Many species of Mollicutes can stably maintain episomal DNA containing the oriC sequence of the same species or a closely related one 19 , 35 , 43 , 44 , 46 , 63 – 65 . For certain species such as M. agalactiae , it has been found that the dnaA gene present in these oriC plasmids is not essential for replication and propagation. The removal of dnaA and simplification of the oriC region results in less frequent integration events at the chromosomal oriC locus in most species 19 , 46 , with the cost of usually lower transformation efficiency rates. On the contrary, in the case of oriC -plasmids derived from Mferi , removal of the dnaA gene resulted in 5 times higher transformation efficiencies regardless of the antibiotic marker used. Only one oriC -plasmid developed in this work, pIVB04, in which the tetM marker had been replaced with pac marker did not yield any transformants despite having the same configuration as pIVB03 (Fig. 1 ). It was only when the orientation of the pac marker was flipped (plasmid pIVB06) that the plasmid yielded a similar number of transformants than pIVB03. This fact suggests that the oriC region contains promoter sequences that could challenge the transcription of the pS' pac cassette by antisense inhibition of gene expression. Antisense RNA-mediated transcriptional attenuation in bacteria is well described 66 – 68 , involving either dsRNA-specific RNases, peptide nucleic acids, phosphorodiamidate morpholino oligomers or just by steric hindrance of transcription or translation. In Mollicutes , antisense RNAs have been identified in pathogenic species of swine such as M. hyopneumoniae 69 and human species like M. pneumoniae 70 or M. genitalium 25 , and their role in modulation of gene expression has been acknowledged. However, it seems likely that by steric hindrance, the RNA polymerase complex cannot successfully read through two genes with colliding orientations if both promoters are spatially close, which should result in a lower expression of both transcripts. This was shown in M. genitalium , when transposons expressing the toxic MG_428 gene coding for the alternative sigma factor σ 20 were all inserted in highly expressed genes in the opposite orientation of transcription, which dampened expression of the toxic gene and allowed cells to live 24 . In the case of pIVB04, most likely the promoter of the dnaN gene is interfering with the expression of the pac marker controlled by the spiralin promoter pS', interfering with the expression of the antibiotic cassette and limiting the puromycin resistance of transformed cells with the oriC -plasmid. This is not the case for pIVB03 and pMYCO1 oriC -plasmids, likely due to the larger size of the tetM marker (1.9kb) compared to the pac cassette (0.6kb). According to the Mferi IVB14/OD_0535 genomic sequence, this strain carries four MIB-MIP copies clustered together in the same genetic locus. Our transcriptomics analyses showed that each pair is an individual transcriptional unit - an operon - with its own putative promoter and a short palindromic sequence resembling rho-independent terminator sequences. We showed that the promoter of the first and last gene tandems are significantly stronger than the other two, which may influence the transcription of downstream elements. The first promoter element may overcome the terminator element and drive the expression of other MIB-MIP pairs, while the last promoter likely plays a role on the expression of the highly conserved putative ATP-synthase gene cluster situated immediately downstream. It is still not clear if MIB-MIP-related IgG cleavage requires the activity of the downstream ATPase, but our results show that the two gene clusters do not necessarily work in cis . In keeping with this, the MIB-MIP systems present in M. hyopneumoniae are unlinked to the ATPase gene cluster (Fig. 6 A and Supp. Figure S4), which is still present but in a different genetic context in this species. Due to their reduced-size genomes, Mollicutes are thought to be devoid of many transcriptional regulatory elements, and rho-independent terminators have been suggested as major fine-tuning, transcription-controlling elements 71 , in conjunction with DNA supercoiling and RNA degradation 72 . We could demonstrate that the promoter elements of the first MIB-MIP gene copy of Mferi (P MM1mfe ) was capable of successfully drive the expression of all MIB-MIP gene tandems individually in trans , which makes it a promising tool for recombinant expression of other rather large membrane proteins using this bacterium in the future. Despite several attempts, expression of active heterologous MIB-MIP systems could only be achieved with a MIB-MIP system of Mmc , which is phylogenetically closely related to Mferi . Expression of functional MIB-MIP gene tandems from the porcine Mollicutes species M. hyopneumoniae and M. hyorhinis was not possible, despite the use of native promoters from Mferi or adapting the codon usage. The structure of the MIB-MIP tandem system has been recently obtained and characterized 9 and shows direct contacts between the two protein counterparts and with the targeted immunoglobulin. Therefore, correct export and folding of both proteins is likely pivotal for the system to work. We show that IgG cleavage of M. hyopneumoniae and M. hyorhinis in cellulo is possible under the standard laboratory conditions, which indicates that the systems present in these bacteria are active when expressed correctly. Despite our efforts, we failed at expressing active MIB-MIP systems from distantly related Mollicutes in Mferi , most likely due to problems related to the export of the system to the membrane. Despite many years of research, the protein export systems of Mollicutes are poorly characterized 73 . Most MIP proteins analyzed in this work contain a classic lipoprotein signal peptide (type II signal peptide), which consist of a positively charged N-terminal region, followed by a central hydrophobic area and a polar C-terminal region (lipobox) 74 . This signal peptide is also known to contain a preserved motif LXXC, which is recognized by the preprolipoprotein diacylglyceryl transferase (Lgt, encoded in MF5583_00077 and 00079 in Mferi IVB14/OD_0535) followed by the apolipoprotein N- acyltransferase (Lnt, encoded in MF5583_00341) that will create the linkage of the protein to the cell membrane after translocation via the Sec pathway 75 . However, none of the MIB proteins analyzed have a similar type II signal peptide or any clear transmembrane domain that suggests in silico association at the cell surface (Supp. Fig. S7), aside from the interaction with MIP required for immunoglobulin cleavage. Furthermore, very low MIB protein levels are detected in our proteomics analyses in either Mmc or Mferi , as it was previously reported in Mmc 5 . In addition, in another closely related Mycoplasma species namely Mmm , only the MIP proteins have been clearly detected in the surface proteome 76 . Similarly, the closely related protein M, present in other Mollicutes species usually devoid of MIB-MIP systems 5 , also lacks any clear membrane anchoring signal and could not be identified in the protein membrane enriched fractions or cell-surface protein labelling in a thorough proteomics study carried out in M. genitalium 77 . However, a recent study characterizing the protein M homolog (IbpM) from Mycoplasmoides pneumoniae shows data indicating that this protein is located at the cell surface 78 , despite that advanced transmembrane domain predictors like DeepTMHMM 79 do not predict the presence of any transmembrane domain in neither protein M nor MIB. Understanding how these and other proteins lacking conventional signal peptides are exported in Mferi is crucial to develop functional display systems in this bacterium. In conclusion, in this work we assessed pathogenicity of Mferi in an established animal model, developed new oriC-based vectors for rapid and versatile gene delivery in this microorganism and use them to characterize expression of native and foreign anti-immunoglobulin systems of Mollicutes , providing new data regarding the molecular mechanisms of these specialized machineries that should aid in the understanding of the immune evasion strategies of pathogenic Mollicutes species. Moreover, we identified promotors suitable to drive expression of rather large heterologous surface proteins, which will be pivotal for future applications of Mferi as a bacterial vaccine chassis. Declarations Author contributions JJ and ST-P conceived the project and planned the experiments. ST-P, SC-P, FL and TY carried out the laboratory experimental work. ST-P and VC prepared the samples for -omics analyses. HA and PK performed the bioinformatics analyses. HP, NR, JJ and TD carried out the animal experimentation or analyzed the clinical data. ST-P and JJ drafted the manuscript. All authors read and agreed to the final version of the manuscript. Acknowledgements This work was supported by the Swiss National Science Foundation (grant number 310030_201152, www.snf.ch ). The in vivo experiment was supported by the International Development Research Centre (Grant ID:108625). We thank the Lausanne Genomic Technologies Facility (University of Lausanne) for genome sequencing of the strains used in this work. We thank the Core Facility Proteomics & Mass Spectrometry (University of Bern) for their assistance in the proteomics analyses. We are grateful to the staff of the Institute for Virology and Immunology (IVI) for their assistance with the animal experimentation. We thank Isabelle Brodard and Bettina Trüeb for their assistance. References Namba, S.: Molecular and biological properties of phytoplasmas. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3854399","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267457955,"identity":"30f96084-5224-4113-8611-45e2c461b9bb","order_by":0,"name":"Sergi Torres-Puig","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYLCCBARTgoEfSB4GMQ3wa4FLSzBINhCjBUXa4AADAzM+LfLtvY9fPGD4I2c++/Dhz4VtFnabb+QePFxQwSBvjsv4M8fNLIAOM5Y5l5YmPbNNInnbjbyEwzPOMBjubMChRSKNzQCoJXEGD48ZMy9Qi9mNHIPDvG0MCSAXYnXY/GcwLfyfP4O0GM8AafmHWwvDDTbmB1BbGKSBWuwMJEBaGnBrMTiTxgaUNTaW4GEzk55xTiJB4swbg8MzjkkYbsDlsPZjzB9/VMjJSfAwP/5cUFZnz9+eY/y5oMZGHqfDGBjYJGBRAIqOxAYIWwKnepDCD3AWENvjUzoKRsEoGAUjEwAAM2lT0EAr5QEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8976-6488","institution":"University of Bern","correspondingAuthor":true,"prefix":"","firstName":"Sergi","middleName":"","lastName":"Torres-Puig","suffix":""},{"id":267457956,"identity":"b93ca508-7ddc-4250-97bd-62c9a8eb3811","order_by":1,"name":"Silvia Crespo-Pomar","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Crespo-Pomar","suffix":""},{"id":267457957,"identity":"66de0f82-6ad5-4f5c-a203-f8311dde6c0a","order_by":2,"name":"Hatice Akarsu","email":"","orcid":"","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Hatice","middleName":"","lastName":"Akarsu","suffix":""},{"id":267457958,"identity":"479889bb-e977-46ce-84b1-ba7624b79a3f","order_by":3,"name":"Thatcha Yimthin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thatcha","middleName":"","lastName":"Yimthin","suffix":""},{"id":267457959,"identity":"ac609100-3ea9-4442-8b99-242bf3ee6704","order_by":4,"name":"Valentina Cippà","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Cippà","suffix":""},{"id":267457960,"identity":"39aec374-fc0d-4ce9-9a4c-bcf1aea536f6","order_by":5,"name":"Thomas Démoulins","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Démoulins","suffix":""},{"id":267457961,"identity":"33864820-15a1-45de-adb7-d19927e38458","order_by":6,"name":"Horst Posthaus","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Horst","middleName":"","lastName":"Posthaus","suffix":""},{"id":267457962,"identity":"6142ca97-3118-4fe8-ab23-5385b66c76f7","order_by":7,"name":"Nicolas Ruggli","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Ruggli","suffix":""},{"id":267457963,"identity":"bb9add83-ef46-4d70-9443-ebd51ee2f8a6","order_by":8,"name":"Peter Kuhnert","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Kuhnert","suffix":""},{"id":267457964,"identity":"6784aff0-d43c-4525-a9f5-2a6d4baa3aab","order_by":9,"name":"Fabien Labroussaa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fabien","middleName":"","lastName":"Labroussaa","suffix":""},{"id":267457965,"identity":"71a3ceb8-bb17-4294-ad38-4a1fcaf02e41","order_by":10,"name":"Jörg Jores","email":"","orcid":"https://orcid.org/0000-0003-3790-5746","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Jores","suffix":""}],"badges":[],"createdAt":"2024-01-11 18:30:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3854399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3854399/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-024-06497-8","type":"published","date":"2024-06-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50389109,"identity":"b872e06b-ab35-475e-aaa8-dfc387ee3ee3","added_by":"auto","created_at":"2024-01-30 18:23:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of replicative \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eoriC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-plasmids for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. feriruminatoris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A) \u003c/strong\u003eOverview of the oriC- plasmids designed in this work. \u003cstrong\u003eB) \u003c/strong\u003eTransformation efficiency upon introduction of the different shuttle vectors in \u003cem\u003eM. feriruminatoris\u003c/em\u003e. A total of four independent biological replicates were conducted. Transformants with pIVB03 and pIVB08 were selected with 15 µg mL\u003csup\u003e-1\u003c/sup\u003e tetracycline, while 16 µg mL\u003csup\u003e-1\u003c/sup\u003e puromycin was used to select transformants with pIVB04, pIVB06 and pIVB09. \u0026nbsp;Data was analyzed using one-way ANOVA tests with Tukey’s multiple comparisons test. ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep\u0026lt;\u003c/em\u003e0.001, n.s. non-significant.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/991ae9d59276384a116c3116.png"},{"id":50389111,"identity":"2d150d9a-176d-4943-b9cf-69c7ff0dc004","added_by":"auto","created_at":"2024-01-30 18:23:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of paired MIB-MIP systems in different \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMollicutes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e species. A)\u003c/strong\u003e Unrooted phylogenetic tree representing concatenated MIB-MIP protein pairs in representative strains of different species of \u003cem\u003eMollicutes\u003c/em\u003e. Colors indicate the different phylogenetic groups: \u003cem\u003eMycoplasma\u003c/em\u003e (in dark red), \u003cem\u003eMycoplasmopsis\u003c/em\u003e(in green), \u003cem\u003eMetamycoplasma \u003c/em\u003e(in dark yellow), \u003cem\u003eUreaplasma\u003c/em\u003e (in purple), \u003cem\u003eMesomycoplasma\u003c/em\u003e (in blue) and\u003cem\u003e Mycoplasmoides\u003c/em\u003e (in turquoise). Animal hosts of the different species are also displayed. The most distant branch is highlighted in grey. More information regarding strains and proteins displayed in the tree can be found in Supp. Table S1. \u003cstrong\u003eB) \u003c/strong\u003eRooted phylogenetic tree showcasing the phylogenetic distance of representative strains of \u003cem\u003eMycoplasmatota \u003c/em\u003eaccording to the 16S RNA. \u003cem\u003eClostridium innocuum \u003c/em\u003eis displayed as an outgroup. Colors are used to distinguish between different phylogenetic groups as in A).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/cee54d7ad01359a6374e8aef.png"},{"id":50389107,"identity":"6c088a12-38c7-4be9-8e67-ef73e4650b62","added_by":"auto","created_at":"2024-01-30 18:23:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of MIB-MIP gene pair expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMferi \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eusing -omics. A) \u003c/strong\u003eTranscriptional analysis using RNAseq of three biological replicates. Top graph shows the mean fragments per kilobase of transcript per million mapped reads (FPKM) of each gene of the MIB-MIP cluster. Bottom graph indicates the read coverage obtained in each replicate. \u003cstrong\u003eB) \u003c/strong\u003eTotal gene distribution based on gene expression measured by RNAseq. All MIB-MIP partners have similar expression levels. \u003cstrong\u003eC) \u003c/strong\u003eTotal protein distribution based on protein expression measured by mass spectrometry.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/d48400431e6a5d3c710973eb.png"},{"id":50389737,"identity":"de1fe88e-02ec-442f-96ea-d3360cc9bee4","added_by":"auto","created_at":"2024-01-30 18:31:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the operonic structure of the MIB-MIP gene cluster. A) \u003c/strong\u003eSchematic organization of the chromosomal locus where the MIB-MIP gene cluster is located in \u003cem\u003eM. feriruminatoris\u003c/em\u003e IVB14/OD_0535. Small arrows depict promoter locations, while hairpins indicate the presence of putative terminator sequences. Graph shows the fragment analysis obtained after primer extension of the MIB4 gene transcript, with a major peak at 478nt upstream of the primer binding site. On the right of the graph, the predicted -35 box, Pribnow box and experimentally determined transcriptional start site (TSS) of the MIB-MIP4 are highlighted. \u003cstrong\u003eB)\u003c/strong\u003e Presence of large, inverted repeats after the MIP genes of several species of the ‘\u003cem\u003eMycoplasma mycoides\u003c/em\u003e cluster’. First MIB-MIP operon of \u003cem\u003eMmm\u003c/em\u003e strain Gladysdale is interrupted by an insertion sequence. \u003cem\u003eMmm \u003c/em\u003estrain Gladysdale and \u003cem\u003eM. leachii\u003c/em\u003e strain PG50 only have three MIB-MIP gene copies, therefore the last inverted repeat is absent. \u003cstrong\u003eC) \u003c/strong\u003eConsensus sequence of the inverted repeat built with WebLogo tool. Sequence can form a hairpin loop with a minimum free energy (MFE) of -12.70 kcal/mol, as calculated with RNAfold webserver.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/4614752d2472144ed20d852f.png"},{"id":50389114,"identity":"c49e0934-4b3f-43e6-ae48-3136c04c331c","added_by":"auto","created_at":"2024-01-30 18:23:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and characterization of a MIB-MIP deficient \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMferi \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrain. A)\u003c/strong\u003e Schematic representation of the MIB-MIP operons and their genomic context in the ruminant mycoplasmas \u003cem\u003eMmc\u003c/em\u003e GM12 and \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535. Deletion of the four MIB-MIP gene pairs of \u003cem\u003eMferi \u003c/em\u003eresults in the ΔMIB/MIP strain, in which these genes (~20Kb) have been replaced by a tetracycline resistance cassette (PS’\u003cem\u003etetM\u003c/em\u003e) and yeast replication elements (YRE) (~4.1Kb). \u003cstrong\u003eB) \u003c/strong\u003eGrowth curve determined by CFU mL\u003csup\u003e-1\u003c/sup\u003e of three independent biological replicates of cultivated \u003cem\u003eMmc \u003c/em\u003eGM12 (in red), \u003cem\u003eMferi \u003c/em\u003eIVB14/OD_0535 (in blue) and \u003cem\u003eMferi \u003c/em\u003eΔMIB/MIP strain (in green). pH of the cultures at certain time-points is indicated. Note the rapid decline of cell titers upon medium acidification by \u003cem\u003eMferi\u003c/em\u003e. \u003cstrong\u003eC\u003c/strong\u003e) Doubling time calculated during exponential phase of the three strains growing in standard SP-5 medium at 37° C without antibiotic selection. Results are the average of 4 biological replicates. \u003cem\u003ep\u0026lt; \u003c/em\u003e0.05. \u003cstrong\u003eD)\u003c/strong\u003e IgG cleavage activity of several strains of \u003cem\u003eMferi \u003c/em\u003edetermined by Western blot. Only mutant strains carrying the plasmids pIVB09_MM1mfe and pIVB09_MM4mfe can restore IgG cleavage activity. Cleaved IgG heavy chain is indicated with an arrow. \u003cstrong\u003eE) \u003c/strong\u003eIgG cleavage activity in a MIB-MIP mutant strain can be restored with MIB-MIP pairs 2 and 3 if they are under the control of the P\u003csub\u003eMMMfe1\u003c/sub\u003e promoter, measured by Western blot.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/b258e04d2d4fecf311795ea3.png"},{"id":50389738,"identity":"0e111219-4512-4e12-bfff-ac74873fee22","added_by":"auto","created_at":"2024-01-30 18:31:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":311162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIgG cleavage activity in porcine \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMesomycoplasma \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003especies. A) \u003c/strong\u003eSchematic representation of the chromosomal location of the MIB-MIP systems of \u003cem\u003eM. hyopneumoniae\u003c/em\u003e and \u003cem\u003eM. hyorhinis\u003c/em\u003e. \u003cstrong\u003eB)\u003c/strong\u003ePorcine IgG cleavage activity of \u003cem\u003eM. hyopneumoniae\u003c/em\u003e and \u003cem\u003eM. hyorhinis \u003c/em\u003eanalyzed by Western blot. \u003cstrong\u003eC) \u003c/strong\u003ePorcine and goat IgG cleavage activity of \u003cem\u003eMferi \u003c/em\u003eΔMIB/MIP strain expressing heterologous MIB-MIP systems.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/f31b36e70e111d63ccfbdf34.png"},{"id":50390006,"identity":"48c575b1-7c89-4aa0-9dbf-daddcfa15f03","added_by":"auto","created_at":"2024-01-30 18:39:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":201928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous MIB-MIP expression analysis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMferi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A) \u003c/strong\u003eSchematic representation of three plasmids carrying tagged versions of MIB-MIP copies from different species (top). Detection of 6xHis and FLAG-tagged MIB and MIP, respectively. DnaK was used as a loading control (bottom). \u003cstrong\u003eB)\u003c/strong\u003e Schematic representation of the two plasmids used to assess the role of the signal peptides in the expression of the MIB-MIP system of \u003cem\u003eM. hyorhinis\u003c/em\u003e (top). Detection of 6xHis and FLAG-tagged MIB and MIP, respectively. DnaK was used as a loading control (bottom).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/573ace94bcf2edf71c495a1b.png"},{"id":50389736,"identity":"15e79fad-efdc-48eb-9fd5-8986bc96c7e2","added_by":"auto","created_at":"2024-01-30 18:31:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":26209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonitoring of body temperature during the animal challenge. \u003c/strong\u003eTwo groups of three outbreed goats were infected with either \u003cem\u003eM. capricolum \u003c/em\u003esubsp. \u003cem\u003ecapripneumoniae\u003c/em\u003eILRI181 (in red) or \u003cem\u003eM. feriruminatoris \u003c/em\u003eG5847 (in blue). Body temperature was monitored daily for both groups until experiment termination. Goats infected with \u003cem\u003eMccp\u003c/em\u003e were euthanized at day 10 due to the severity of their clinical signs, as indicated with a cross.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/3e140c30bce729de3c5fd9d0.png"},{"id":59310915,"identity":"7a66e9e6-93ec-4809-a81c-c2d99ab8ac98","added_by":"auto","created_at":"2024-06-29 07:07:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2319185,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/f5d57767-d21b-4aa8-a3d4-322691c5716d.pdf"},{"id":50389115,"identity":"ab782994-df54-4750-b21e-cb3c5682c82a","added_by":"auto","created_at":"2024-01-30 18:23:40","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3702179,"visible":true,"origin":"","legend":"","description":"","filename":"SuppMatAll.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3854399/v1/9da815fdadfaf45f7c22a774.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Characterization of the MIB-MIP system of different Mollicutes using an engineered \u003ci\u003eMycoplasma feriruminatoris\u003c/i\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacteria of the class \u003cem\u003eMollicutes\u003c/em\u003e are characterized by the absence of a cell wall and numerous enzymatic pathways that were lost by reductive evolution from Gram-positive ancestors. As a result, \u003cem\u003eMollicutes\u003c/em\u003e have a pleomorphic cell shape and live a parasitic lifestyle to scavenge nutrients from their host. A number of \u003cem\u003eMollicutes\u003c/em\u003e infecting plants or animals including humans are pathogenic, such as the well-known pathogens \u003cem\u003eCandidatus Phytoplasma asteris\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, members of the \u0026ldquo;\u003cem\u003eMycoplasma mycoides\u003c/em\u003e cluster\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eMycoplasmoides pneumoniae\u003c/em\u003e and \u003cem\u003eMycoplasmoides genitalium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, respectively. Knowledge of their virulence traits is still scarce due to the fastidious nature of these organisms and the historical lack of genetic tools to modify their genomes. Recently, the candidate virulence factor Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system has been characterized in various \u003cem\u003eMollicutes\u003c/em\u003e species \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This system consists of at least two surface located proteins that bind and cleave the variable region of the heavy chain (Vh) of IgGs and has been shown to be active \u003cem\u003ein vivo\u003c/em\u003e in goats infected with \u003cem\u003eMycoplasma mycoides\u003c/em\u003e subsp. \u003cem\u003ecapri\u003c/em\u003e (\u003cem\u003eMmc\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Cryo-electron microscopy visualized how the two proteins bind to the Fab fragment in a \u0026ldquo;hug of death\u0026rdquo; mechanism, which is thought to interfere with antibody-antigen interactions\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycoplasma feriruminatoris\u003c/em\u003e (\u003cem\u003eMferi\u003c/em\u003e) is a close relative of the \u003cem\u003eMollicutes\u003c/em\u003e belonging to the '\u003cem\u003eM. mycoides\u003c/em\u003e cluster' and has been isolated from Alpine ibex and Rocky Mountain goats \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This species is characterized by its short doubling time compared to the slow growth typically observed in many other \u003cem\u003eMollicutes\u003c/em\u003e. The genome of \u003cem\u003eMferi\u003c/em\u003e has been recently adapted to synthetic genomics techniques \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, including genome editing in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and genome transplantation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. All these features together with the simplistic nature of \u003cem\u003eMollicutes\u003c/em\u003e, absence of a cell wall and different genetic code have turned \u003cem\u003eMferi\u003c/em\u003e and other \u003cem\u003eMollicutes\u003c/em\u003e as a promising candidate to serve as a workhorse for industrial applications \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e such as \u003cem\u003ein vivo\u003c/em\u003e vaccine- or drug delivery \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this work we tested the pathogenicity of \u003cem\u003eMferi\u003c/em\u003e in domestic goats \u003cem\u003ein vivo\u003c/em\u003e and we developed \u003cem\u003eoriC\u003c/em\u003e-based plasmids to allow rapid introduction of genes and \u003cem\u003ein cellulo\u003c/em\u003e expression of homologous and heterologous DNA at a high turnaround time. We also analyzed in depth the operons expressing the four MIB-MIP gene copies present in \u003cem\u003eMferi\u003c/em\u003e and multiple other \u003cem\u003eMollicutes\u003c/em\u003e species and expressed them individually to test their activity, proving that \u003cem\u003eMferi\u003c/em\u003e and the tools developed in this work are valuable for functional genomics of this and other \u003cem\u003eMollicutes\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains used and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e Stellar cells (Clontech) or NEB 5-alpha (New England Biolabs) were used for all constructions of different \u003cem\u003eoriC\u003c/em\u003e-plasmids and subsequent plasmid preparations. All \u003cem\u003eE. coli\u003c/em\u003e strains were cultured in Luria Bertani (LB) medium at 37\u0026ordm; C and shaking at 220 rpm or on LB agar plates supplemented with 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ampicillin when necessary. Transformation of \u003cem\u003eE. coli\u003c/em\u003e strains was achieved by using a heat-shock standard protocol.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. cerevisiae\u003c/em\u003e strain W303a was used to modify and propagate the \u003cem\u003eMferi\u003c/em\u003e genome. \u003cem\u003eS. cerevisiae\u003c/em\u003e was cultured in Yeast Peptone Dextrose Adenine (YPDA) or Synthetic Defined (SD) broth (Formedium) depleted for tryptophan, uracil and/or histidine depending on the auxotrophic marker in use. \u003cem\u003eS. cerevisiae\u003c/em\u003e strains were cultured at 30\u0026deg; C and 220 rpm.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 \u003csup\u003e14\u003c/sup\u003e, \u003cem\u003eMesomycoplasma hyorhinis\u003c/em\u003e JF5820 \u003csup\u003e17\u003c/sup\u003e, and \u003cem\u003eMesomycoplasma hyopneumoniae\u003c/em\u003e Ue273 used in this study were isolated from diagnostic material at the Institute of Veterinary Bacteriology in Bern. \u003cem\u003eM. hyopneumoniae\u003c/em\u003e Ue273 was isolated from bronchial tissue of a Swiss wild board. \u003cem\u003eMferi\u003c/em\u003e strains used for -omics and \u003cem\u003ein vitro\u003c/em\u003e studies were grown at 37\u0026ordm; C with 5% CO\u003csub\u003e2\u003c/sub\u003e in SP-5 medium \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e or modified Hayflick agar plates \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e supplemented with 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tetracycline or 16 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e puromycin when necessary. Presence of \u003cem\u003eoriC\u003c/em\u003e-plasmids in liquid cultures was maintained by puromycin (8 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eM. hyorhinis\u003c/em\u003e and \u003cem\u003eM. hyopneumoniae\u003c/em\u003e were grown in Friis medium at 37\u0026ordm; C. \u003cem\u003eMycoplasma capricolum\u003c/em\u003e subsp. \u003cem\u003ecapricolum\u003c/em\u003e ΔRE (\u003cem\u003eMcap\u003c/em\u003e ΔRE) was used as a recipient for genome transplantation from yeast \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (see \u0026lsquo;Genome transplantation\u0026rsquo; section below). For the experimental infection \u003cem\u003eM. capricolum\u003c/em\u003e subsp. \u003cem\u003ecapripneumoniae\u003c/em\u003e (\u003cem\u003eMccp\u003c/em\u003e) ILRI181 and \u003cem\u003eMferi\u003c/em\u003e G5847\u003csup\u003eT\u003c/sup\u003e were grown at 37\u0026deg; C with 5% CO\u003csub\u003e2\u003c/sub\u003e in Mycoplasma Experience Liquid Medium (Mycoplasma Experience), aliquoted and stored at -80\u0026deg; C until used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe phylogenetic analysis based on the 16S rRNA gene sequences of the \u003cem\u003eMollicutes\u003c/em\u003e covered overall \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 genera, encompassing \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;27 species and \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36 strains. The tree was built in BioNumerics v8.1 using Jukes-Cantor correction and the Neighbour Joining method. \u003cem\u003eClostridium innocuum\u003c/em\u003e was used as outgroup.\u003c/p\u003e \u003cp\u003eFor the phylogenetic analysis of the MIB-MIP system the translated amino acid sequences were used using the genomes described above (Supp. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Sequences were trimmed to a uniform size and used for analysis. Orphan copies of either the MIB or MIP were not included in the analysis. We compared the trees based on the MIB, MIP or MIB-MIP using a Pearson correlation. Unrooted phylogenetic trees were build using the model LG\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4. The trees were then plotted and manually edited in FigTree (v1.4.4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA extraction and Next Generation Sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA (gDNA) from \u003cem\u003eMollicutes\u003c/em\u003e was extracted from 20 mL cultures using the Promega Wizard Genomic DNA purification kit. The quality and quantity of the gDNA was assessed on agarose gels and using the Qubit fluorometer (Invitrogen). Subsequently, gDNA was sequenced in the PacBio sequencing platform at the Lausanne Genomic Technologies Facility at the Center for Integrative Genomics, University of Lausanne as described elsewhere (Hill et al., 2021). Briefly, DNA was sheared in a Covaris g-TUBE (Covaris, Woburn, MA, USA) to obtain 10 kb fragments and the DNA size distribution was confirmed on a Fragment Analyzer (Advanced Analytical Technologies, Ames, IA, USA). A barcoded SMRTbell library was prepared with 480 ng of gDNA using the PacBio SMRTbell Template Express Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA), according to the manufacturer's recommendations. Libraries were pooled and sequenced with v3.0/v3.0 chemistry on a PacBio Sequel instrument (Pacific Biosciences, Menlo Park, CA, USA) at 10 hours movie time, pre-extension time of 2 hours, using one SMRT cell v3. Genomes were assembled from PacBio reads using the software Flye, version 2.6 \u003csup\u003e21\u003c/sup\u003e. Circularized genomes were polished with three rounds with the Arrow software [single-molecule real-time (SMRT) Link version 8 package]. Genomes were rotated to the first nucleotide of the start codon of the \u003cem\u003ednaA\u003c/em\u003e gene, and annotated using Prokka, version 1.13 \u003csup\u003e22\u003c/sup\u003e. \u003cem\u003eM. hyopneumoniae\u003c/em\u003e Ue273, \u003cem\u003eMesomycoplasma ovipneumoniae\u003c/em\u003e 14KM848 and \u003cem\u003eMferi\u003c/em\u003e ΔMIB-MIP sequences are deposited as BioProject PRJNA1062711.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic analysis and primer extension\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMferi\u003c/em\u003e for transcriptomics analysis was carried out as previously described \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Briefly, RNA from three biological replicates of \u003cem\u003eMferi\u003c/em\u003e was extracted from liquid culture using the Zymo Research Quick-RNA Fungal/Bacterial Miniprep kit. RNA quality was assessed at the Lausanne sequencing platform on a Fragment Analyzer (Agilent Technologies). Libraries were prepared using the Illumina TruSeq Stranded mRNA reagents (Illumina), excluding the polyA selection step and using a unique dual indexing strategy. Ribosomal rRNA depletion was carried out with QIAseq FastSelect\u0026ndash;5S/16S/23S kit (Qiagen). Libraries were quantified by QubIT, Life Technologies and their quality was assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 1.92 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 2 x 150 cycles (paired end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (v. 2.20, Illumina).\u003c/p\u003e \u003cp\u003eFor primer extension analyses, total RNA was extracted from 10mL culture at early stationary phase using the RNAqueous Total RNA Isolation kit (ThermoFisher Scientific) following manufacturer's instructions. Primer extension reactions were performed using 20\u0026ndash;25\u0026micro;g of total RNA, the SuperScript IV First Strand Synthesis system (ThermoFisher Scientific) and 6-Carboxyfluorescein (6-FAM) labeled primers (Supp. Table S2), as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e with few modifications. Briefly, 20\u0026ndash;25\u0026micro;g of total RNA were mixed with 0.5\u0026micro;L of 5\u0026micro;M 6-FAM labelled primer and 1.5\u0026micro;L of dNTPs in a 20\u0026micro;L final volume reaction. This mix was incubated for 5 min at 65\u0026ordm;C in a thermocycler and subsequently cooled down on ice. In a separate tube, a 10\u0026micro;L reaction mix was prepared by adding 6\u0026micro;L 5x SuperScript IV RT buffer, 1.5\u0026micro;L 0.1M DTT, 60U of RNase Inhibitor, and 1\u0026micro;L of SuperScript IV Reverse Transcriptase. This reaction mix was added to the tube containing the RNA and primer on ice and mixed by gently pipetting before incubating at 55\u0026ordm;C for 30 min in a thermocycler. The reaction was inactivated at 80\u0026ordm;C for 10 min prior addition of 1\u0026micro;L RNase H and incubation at 37\u0026deg;C for 1h. cDNA was precipitated with 0.1 volumes of 3M sodium acetate and 2.5 volumes of absolute ethanol. The cDNA pellet was washed in 70% ethanol, resuspended in 10\u0026micro;L Hi-Di formamide (ThermoFisher Scientific) and kept protected from light exposure at room temperature. Samples were separated and analyzed in an ABI3730XL instrument at Microsynth AG (Switzerland) using ROX size standards.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProteomics analysis\u003c/h2\u003e \u003cp\u003eProteomics analyses were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Briefly, the same three biological replicates of mycoplasmas used for transcriptomics were harvested by centrifugation at 4,000 x g at 4\u0026deg;C for 15min. Pellets were washed three times in ice-cold PBS and stored at -80\u0026deg;C until further use. The cells pellets were thawed on ice and subsequently lysed via resuspension in 8M urea/ 100mM Tris-HCl and precipitated overnight at -20˚C in acetone. Protein pellets were air-dried at room temperature for 15 min before being reconstituted in 8M urea/50mM Tris-HCl. A protein aliquot corresponding to 10\u0026micro;g protein was trypsinized overnight at room temperature in digestion buffer and subsequently stopped by adding 1% (v/v) tri-fluoroacetic (TFA). Three repetitive injections of aliquots corresponding to 500ng of trypsinized proteins were processed by liquid chromatography (LC)-MS/MS (PROXEON coupled to a QExactive HF mass spectrometer, ThermoFisher Scientific). Mass spectrometry-derived proteomic data were analyzed against custom-made databases by Transproteomic pipeline (TPP) tools \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The four database search engines Comet \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, Xtandem \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, MSGF \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and MyriMatch \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e were used and each search was followed by the application of the PeptideProphet tool \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The iProphet \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e software was subsequently used to summarize the search results, which were filtered at the false discovery rate of 0.01. Protein identifications were exclusively accepted if at least two of the search engines agreed on the identification. The decoy approach was used for custom databases containing standard entries and protein inference was investigated using ProteinProphet. For those protein groups accepted by a false discovery rate filter of 0.01, a Normalized Spectral Abundance Factor (NSAF) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e was calculated based on the peptide to spectrum match count. Shared peptides were considered by a method published elsewhere \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eAll plasmids used in this study were constructed using the NEBuilder HiFi DNA Assembly kit (New England Biolabs), following manufacturer\u0026rsquo;s instructions. The plasmid pIVB03 was constructed by replacing the origin of replication of \u003cem\u003eMmc\u003c/em\u003e GM12 present in pMYCO1 \u003csup\u003e35\u003c/sup\u003e by the origin of replication of \u003cem\u003eMferi\u003c/em\u003e type strain G5847\u003csup\u003eT \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, amplified with primers #001 and #002 (Supp. Table S2). The plasmid pIVB04 is a derivate of pIVB3 in which the \u003cem\u003etetM\u003c/em\u003e marker under the control of the spiralin promotor (pS\u0026rsquo;) was replaced by pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker, amplified from the gDNA of \u003cem\u003eMcap\u003c/em\u003e ΔRE strain \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e using primers #003 and #004. The plasmid pIVB06 is a derivate of pIVB04 in which the orientation of the pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker was switched. This switch was performed by amplifying the pIVB04 backbone without the pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker using primers #007 and #008 and the pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker using primers #005 and #006. Plasmid pIVB08 is a derivate of plasmid pIVB03 and was constructed by assembling the pS\u0026rsquo;\u003cem\u003etetM\u003c/em\u003e marker amplified with primers #011 and #012 with the pIVB03 backbone amplified in two parts using primers #009 with #010, and #013 with #014. The plasmid pIVB09 was built by replacing the pS\u0026rsquo;\u003cem\u003etetM\u003c/em\u003e cassette from pIVB08 by the pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker, employing primers #009 and #016 for the amplification of the pIVB08 backbone and primers #011 and #015 to amplify the pS\u0026rsquo;\u003cem\u003epac\u003c/em\u003e marker.\u003c/p\u003e \u003cp\u003eAll plasmids carrying MIB-MIP copies of the different \u003cem\u003eMollicutes\u003c/em\u003e have the pIVB09 as a backbone, amplified using primers #017 and #018. The MIB-MIP gene pairs and their natural promoter regions were amplified from gDNA of each respective host strain (\u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535, \u003cem\u003eM. hyopneumoniae\u003c/em\u003e Ue273, \u003cem\u003eM. hyorhinis\u003c/em\u003e JF5820) using primers listed in Supp. Table S2. To generate plasmids expressing MIB-MIP gene copies with the promoter region of the first MIB-MIP gene copy of \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eMM1mfe\u003c/em\u003e\u003c/sub\u003e), the pIVB09 backbone containing the \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eMM1mfe\u003c/em\u003e\u003c/sub\u003e was amplified using primers #017 and #051 and each MIB-MIP copy with their respective primer set listed in Supp. Table S2. Plasmid carrying the tagged MIB-MIP copy number 4 from \u003cem\u003eMmc\u003c/em\u003e was created amplifying the promoter sequence of the first MIB-MIP copy of \u003cem\u003eMmc\u003c/em\u003e using primers #057 and #058, and each gene with primers #059 and #060, and #061 and #062. Plasmids bearing codon-optimized MIB-MIP gene pairs tagged with 6xHis and FLAG, respectively, were created by cloning each gene copy, which was custom synthesized (GenScript). Optimization was carried out by using the Optimizer tool \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. All plasmids generated in this study are listed in Supp. Table S3 and the proper manipulations were sequence verified by Sanger sequencing at Microsynth AG (Switzerland).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCReasPy-Cloning of\u003c/b\u003e \u003cb\u003eM. feriruminatoris\u003c/b\u003e \u003cb\u003egenome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA mutant strain of \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 without the four MIB-MIB gene pairs (ΔMIB-MIP) was generated using the CReasPy-Cloning method \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Briefly, two different gRNA sequences were designed by using the \u0026ldquo;CRISPR Guides\u0026rdquo; tool available in the Benchling work environment (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://benchling.com\u003c/span\u003e\u003cspan address=\"https://benchling.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Target sequences with the highest on-target score and the lowest off-target score were selected. Two gRNAs were designed to target the genes coding for the 4 copies of the MIB-MIP system in \u003cem\u003eMferi\u003c/em\u003e (locus_tags MF5583_00301 - MF5583_00308), by using complementary primers (#084\u0026ndash;087). The corresponding pgRNA plasmids (pgRNA-1MIBMIP, pgRNA-2MIBMIP) were constructed following the protocol described elsewhere \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Plasmids were sequence verified using Sanger sequencing at Microsynth AG (Switzerland) and purified using QIAprep Miniprep Spin Kit (Qiagen). Plasmids were then transformed into \u003cem\u003eS. cerevisiae\u003c/em\u003e W303a-eSpCas9 via lithium acetate transformation \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and transformants were selected on SD-Trp-Ura medium (Takara). Recombination templates containing the yeast elements (YCp1.1) and the tetracycline resistance cassette (pS'\u003cem\u003etetM\u003c/em\u003e) were produced by PCR amplification of the ARS4/CEN6/HIS/pS\u0026rsquo;\u003cem\u003etetM\u003c/em\u003e loci from the plasmid pMT85\u0026ndash;PSTetM-ARSCenHis-pRS313 \u003csup\u003e18\u003c/sup\u003e using primers #082 and #083 with the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and purified using the High Pure PCR Product Purification Kit (Roche). The resulting recombination template contained sequences with 50bp homology to the regions flanking the MIB-MIP locus of the \u003cem\u003eMferi\u003c/em\u003e genome. All primers are listed in Supp. Table S2. \u003cem\u003eMferi\u003c/em\u003e genomes were isolated and introduced in \u003cem\u003eS. cerevisiae\u003c/em\u003e W303a-eSpCas9 pgRNA-1MIBMIP or pgRNA-2MIBMIP by spheroplast transformation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome transplantation into\u003c/b\u003e \u003cb\u003eMcap\u003c/b\u003e \u003cb\u003eΔRE recipient cell\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genome IVB14/OD_0535::YCp-ΔMIB-MIP maintained in \u003cem\u003eS. cerevisiae\u003c/em\u003e as a Yeast Artificial Chromosome (YAC) was transplanted into \u003cem\u003eMcap\u003c/em\u003e ΔRE strain as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, \u003cem\u003eMcap\u003c/em\u003e ΔRE was grown in SOB\u0026thinsp;+\u0026thinsp;medium until early stationary phase (pH 6.5), washed in 10 mM Tris 250 mM NaCl pH 6.5 and resuspended in cold 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e. Transplantation was carried out mixing SP-5 without serum and agarose plugs containing the modified genome with the resuspended cells in 2X Fusion Buffer (20 mM Tris, 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 500 mM NaCl, 10% PEG\u003csub\u003e6000\u003c/sub\u003e pH 6.5). Mixtures were incubated statically for 90 min at 30\u0026deg;C and then plated in SP-5 plates supplemented with 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tetracycline. The resulting transplanted mutant strains were subjected to multiplex and simplex PCRs to confirm integrity of the genome (see primer list in Table S2). Additionally, the genomes of mutant strains were verified via Illumina-type next generation sequencing and mapping assembly to the \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 parental strain \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGrowth rate determination\u003c/h2\u003e \u003cp\u003eGrowth rate of the different mycoplasma strains was assessed by color-changing units (CCU) per mL as well as colony-forming units (CFU) for up to 20 h every 60 min. Briefly, 100 mL SP-5 at pH\u0026thinsp;=\u0026thinsp;7.5 containing 10\u003csup\u003e2\u003c/sup\u003e cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were incubated at 37\u0026ordm; C and 5% CO\u003csub\u003e2\u003c/sub\u003e and small aliquots were removed every hour. Each aliquot was serially diluted and distributed in 200 \u0026micro;L volumes using 96-well plates (for CCU calculation) and plated as spot dilutions in SP-5 agar plates (for CFU calculation). Color change and colonies were assessed after 2 days of incubation. To better compare the growth rate between wild-type (WT) and mutant strain, no additional antibiotics were added to the SP-5 medium during the experiment. Growth curves were plotted, and the growth rates were calculated using GraphPad Prism v9.0.0\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransformation of\u003c/b\u003e \u003cb\u003eM. feriruminatoris\u003c/b\u003e \u003cb\u003eand screening of mutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe transformation protocol used to transform \u003cem\u003eoriC\u003c/em\u003e-plasmids into \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 was adapted from the one used for transformation of \u003cem\u003eMmc\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, with some modifications. \u003cem\u003eMferi\u003c/em\u003e was grown overnight (O/N) in SP-5 with pH adjusted to 8.0 (SP-5\u003csub\u003epH8\u003c/sub\u003e) until late logarithmic phase (~\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;7.0) to achieve the highest total CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (4 mL culture/ transformation). Cells were cooled on ice, pelleted at 4,200 x \u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026ordm; C and washed once in Sucrose/Tris Buffer (0.25 M Sucrose, 10 mM Tris-HCl, pH\u0026thinsp;=\u0026thinsp;7.0). Each cell pellet was resuspended in 400 \u0026micro;L cold CaCl\u003csub\u003e2\u003c/sub\u003e 0.1 M and kept on ice for 30 min. In a 50 mL falcon tube, 10 \u0026micro;L of plasmid (600\u0026ndash;1000 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were added to 400 \u0026micro;L Fusion Buffer 2X (0.5 M Sucrose, 20 mM Tris-HCl, 40% PEG8000, pH\u0026thinsp;=\u0026thinsp;7.0) and left at room temperature. After an incubation of 30 min, the 400 \u0026micro;L cell mix was added into the Falcon tube containing the Fusion Buffer 2X as well as the plasmid and mixed gently. The reaction was left incubating at 30\u0026ordm; C for at least 25\u0026ndash;30 min. Then, fusion was stopped by adding 9 mL of cold SP-5 and inverting the tube once. Cells were recovered by centrifugation at 4,200 x \u003cem\u003eg\u003c/em\u003e for 15 min at 10\u0026ordm; C and the supernatant was carefully discarded. The cell pellet was resuspended in 1 mL fresh SP-5\u003csub\u003epH8\u003c/sub\u003e and incubated at 37\u0026ordm; C for 45\u0026ndash;60 min before plating on selective agar plates. Colonies of transformants were visible after 48 h but were picked up on day 3\u0026ndash;4 after transformation in 1.5 mL microcentrifuge tubes containing 1 mL SP-5\u003csub\u003epH8\u003c/sub\u003e with the appropriate antibiotic (i.e 16 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e puromycin or 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e tetracycline). Transformants were passaged three times before screening. Proper transformants were verified by PCR of cell lysates obtained as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, using the oligonucleotides listed in Supp. Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMIB-MIP-derived IgG cleavage analysis\u003c/h2\u003e \u003cp\u003eIgG cleavage assays were performed as described in \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, with some modifications. For all \u003cem\u003eMferi\u003c/em\u003e strains, cultures were grown O/N in SP-5\u003csub\u003epH8\u003c/sub\u003e with antibiotic selection, if necessary, until stationary phase. Next day, 1mL fresh SP-5\u003csub\u003epH8\u003c/sub\u003e medium was inoculated with 50 \u0026micro;L of the O/N cultures maintaining the antibiotic selection and grown at 37\u0026ordm;C and 5%CO\u003csub\u003e2\u003c/sub\u003e until late logarithmic phase (~\u0026thinsp;10\u003csup\u003e9\u003c/sup\u003e cells). Cells were spun down at 7,000 x \u003cem\u003eg\u003c/em\u003e for 10min, washed once with SP-5 w/o serum \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and spun down again in the same parameters. Pellets were resuspended in 35\u0026micro;L of SP-5 w/o serum containing 250 ng \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of purified IgG from goat or swine serum (Sigma-Aldrich) and incubated at 37\u0026ordm; C for 45 min. Then, cells were pelleted at 7,000 x \u003cem\u003eg\u003c/em\u003e for 10min, and supernatants were recovered and mixed with 7\u0026micro;L 6x Laemmli buffer before boiling at 100\u0026ordm; C for 10 min. Supernatants were separated in a 10% SDS-PAGE and transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (BioRad). Membrane was blocked in PBS with 5% skimmed milk (Becton Dickinson) and 0.05% Tween-20 (Sigma). Antibodies and working dilutions are listed in Supp. Table S4. Membrane was developed using SuperSignal West Pico PLUS Chemiluminiscent substrate (ThermoFisher Scientific), following manufacturer\u0026rsquo;s instructions. In the case of \u003cem\u003eM. hyorhinis\u003c/em\u003e and \u003cem\u003eM. hyopneumoniae\u003c/em\u003e, IgG cleavage determination was performed similarly, but cells were grown in Friis until early stationary phase and no subculture step was performed. Besides, incubation with IgG was extended to 2 h (\u003cem\u003eM. hyorhinis\u003c/em\u003e) or 3 h (\u003cem\u003eM. hyopneumoniae\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of expression of MIB and MIP\u003c/h2\u003e \u003cp\u003eExpression of C-terminal tagged MIB and MIP was analyzed by immunoblot using anti-6xHis and anti-FLAG antibodies (Table S4). Bacterial cells were washed twice in sterile PBS before suspended in 100 \u0026micro;L. Lysates were obtained by adding 20 \u0026micro;L 6x Laemmli buffer and boiling at 100\u0026deg; C for 10 min. Lysates were separated in a 7.5%-8% SDS-PAGE before blotting. The immunoblots were performed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were carried out using GraphPad Prism (v9.0.0). One-way ANOVA tests and Tukey\u0026rsquo;s comparative tests were performed to assess significance and calculate \u003cem\u003ep\u003c/em\u003e values when indicated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003einfection of domestic goats\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe experimental infection of goats was performed in compliance with the Swiss animal protection law (TSchG SR 455; TSchV SR 455.1; TVV SR 455.163) under the cantonal license BE67/19. The experiments were reviewed by the cantonal committee on animal experiments of the canton of Bern, (Switzerland) and approved by the cantonal veterinary authority (Amt f\u0026uuml;r Landwirtschaft und Natur LANAT, Veterin\u0026auml;rdienst VeD, Bern, Switzerland).\u003c/p\u003e \u003cp\u003eThe infection trial was carried out at the Institute of Virology and Immunology (Mittelh\u0026auml;usern, Switzerland) using six female goats in total, aged between 1 and 4 years old and weighting 50\u0026ndash;60 kg and randomly split into two groups of three animals. Animals were infected intranasally with 1 mL 10\u003csup\u003e8\u003c/sup\u003e color changing units mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (CCU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of \u003cem\u003eMferi\u003c/em\u003e strain G5847\u003csup\u003eT\u003c/sup\u003e, or \u003cem\u003eMccp\u003c/em\u003e strain ILRI181 on two consecutive days as described recently \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. One mL of mycoplasma culture was atomized using a 1 mL syringe attached with a MAD Nasal\u0026trade; Intranasal Mucosal Atomization Device (Teleflex, UK) and 500 \u0026micro;L of aerosol was applied to each nostril. At 4 days post-infection (dpi), each animal was infected transtracheally with the same number of color changing units (10\u003csup\u003e8\u003c/sup\u003e CCU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). One mL of culture was followed by flushing with 5 mL sterile phosphate buffered saline. For the duration of the experiment, the animals were housed in a high containment facility, one stable per group of infection. The animals were kept on straw with a local ambient temperature of 20\u0026ndash;22˚ C. Before and after \u003cem\u003eMferi\u003c/em\u003e or \u003cem\u003eMccp\u003c/em\u003e infection, body temperature and clinical status was monitored daily. The clinical status was assessed by a veterinarian, always the same person to ensure unbiased clinical assessment. The rectal body temperature was measured with a digital thermometer, whereas respiratory and heart frequencies employed a stethoscope. Differential blood cell counts were determined from EDTA blood samples using an automated hematology analyzer (VetScan HM5, Abaxis). Animals were euthanized when endpoint criteria were reached or at the end of the trial and subjected to postmortem analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDevelopment of replicative plasmids for\u003c/b\u003e \u003cb\u003eM. feriruminatoris\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genetic tools currently available for manipulation of \u003cem\u003eMferi\u003c/em\u003e are limited and only the introduction and modification of the bacterial genome in yeast has been reported, which is technically challenging and time consuming. To accelerate the shuttle in of genes into \u003cem\u003eMferi\u003c/em\u003e, we decided to generate replicative plasmids based on the origin of replication (\u003cem\u003eoriC\u003c/em\u003e) sequence of the chromosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), as previously reported for other \u003cem\u003eMollicutes\u003c/em\u003e species \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We adapted the oriC-plasmid pMYCO1 for use in \u003cem\u003eMferi\u003c/em\u003e by exchanging the \u003cem\u003eoriC\u003c/em\u003e region of \u003cem\u003eMmc\u003c/em\u003e by the \u003cem\u003eoriC\u003c/em\u003e region of \u003cem\u003eMferi\u003c/em\u003e strain G5847. This first \u003cem\u003eoriC\u003c/em\u003e-plasmid, named pIVB03, could be successfully transformed in \u003cem\u003eMferi\u003c/em\u003e and promoted resistance to tetracycline at 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, as \u003cem\u003eMferi\u003c/em\u003e strains engineered in yeast already have a tetracycline resistance cassette, we developed a derivative plasmid of pIVB03 named pIVB04 carrying the puromycin acetyl transferase (\u003cem\u003epac\u003c/em\u003e) gene, which was reported to confer resistance to puromycin in other closely related \u003cem\u003eMollicutes\u003c/em\u003e species \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This puromycin resistance-conferring plasmid was transformed but did not produce puromycin-resistant colonies carrying pIVB04 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This negative outcome was changed by switching the direction of the \u003cem\u003epac\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which became plasmid pIVB06, and allowed the recovery of resistant colonies at 16 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e puromycin. In other \u003cem\u003eMycoplasma\u003c/em\u003e species, \u003cem\u003eoriC\u003c/em\u003e-plasmids have been shown to be more stable when reducing the length of the \u003cem\u003eoriC\u003c/em\u003e region or by deleting the \u003cem\u003ednaA\u003c/em\u003e gene \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Therefore, we modified the \u003cem\u003eoriC\u003c/em\u003e-plasmids pIVB03 and pIVB06 by replacing the \u003cem\u003ednaA\u003c/em\u003e gene of \u003cem\u003eMferi\u003c/em\u003e by the antibiotic resistance cassette, generating streamlined oriC-plasmids with resistance to tetracycline (pIVB08) or to puromycin (pIVB09) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These plasmid versions transformed with an efficiency up to four times higher than their parental plasmids with \u003cem\u003ednaA\u003c/em\u003e counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). They can be used for subsequent mutant phenotype complementation studies and as versatile backbones for heterologous expression systems reported in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMIB-MIP tandem gene copies are widespread, divergent, and horizontally transferred among the\u003c/b\u003e \u003cb\u003eMollicutes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe MIB-MIP system is widespread between different members of the \u003cem\u003eMollicutes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, but little is known about their diversity. We aimed at studying the occurrence and diversity of the different existing MIB-MIP cleavage systems in different phylogenetic clades of the \u003cem\u003eMollicutes\u003c/em\u003e. Therefore, we selected \u003cem\u003eMollicutes\u003c/em\u003e genomes including the ones reported in this study and analyzed the presence of the MIB-MIP system. Altogether, we included a total of 34 genomes from 23 different species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We analyzed the sequences of a total of 70 genes coding for putative MIB proteins and 71 genes encoding putative MIPs. In most genomes analyzed, both MIB and MIP counterparts were found adjacent in the same genetic locus, most likely forming a single transcriptomic unit. Occasionally we observed \u0026ldquo;orphan\u0026rdquo; MIB-encoding genes without a MIP-encoding gene in its vicinity. However, in those genomes with orphan MIBs, there was always another MIB-MIP pair present elsewhere in the genome, suggesting that Ig cleavage activity can potentially still occur. To facilitate the analysis, only the paired MIB-MIP proteins were included in the construction of the phylogenetic trees and the analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supp. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn initial scrutiny of the phylogenetic relations between MIB-MIP proteins of different species revealed three major distinctive branches, (i) encompassing \u003cem\u003eMycoplasma\u003c/em\u003e and \u003cem\u003eMycoplasmopsis\u003c/em\u003e species infecting ruminants; (ii) \u003cem\u003eUreaplasma\u003c/em\u003e, \u003cem\u003eMetamycoplasma\u003c/em\u003e and some MIB-MIP pairs of porcine \u003cem\u003eMesomycoplasma\u003c/em\u003e species; and (iii) a more distant branch containing the MIB-MIP pairs of \u003cem\u003eMycoplasmoides\u003c/em\u003e and \u003cem\u003eMesomycoplasma\u003c/em\u003e species. Interestingly, we detected several examples of closely related MIB-MIP systems between \u003cem\u003eMollicutes\u003c/em\u003e species that are phylogenetically very distant but share the same hosts, such as \u003cem\u003eUreaplasma\u003c/em\u003e species and \u003cem\u003eMetamycoplasma hominis\u003c/em\u003e, \u003cem\u003eMycoplasmoides gallisepticum\u003c/em\u003e and \u003cem\u003eMycoplasmopsis synoviae\u003c/em\u003e, or \u003cem\u003eMycoplasmopsis pulmonis\u003c/em\u003e and \u003cem\u003eMetamycoplasma arthritidis\u003c/em\u003e, infecting humans, poultry, or rodents, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This data strongly suggests that these genes have been acquired through horizontal gene transfer between distantly related Mollicutes infecting the same host. Species belonging to the \u0026lsquo;\u003cem\u003eM. mycoides\u003c/em\u003e cluster\u0026rsquo; and their close relatives have multiple pairs located adjacent to each other in the same chromosomal locus. Here we show that there is a strong conservation between each pair in the same position, suggesting that multiple copies of this tandem system were present in a common ancestor of these mycoplasmas. Furthermore, there is a significant difference between the two strains of \u003cem\u003eMferi\u003c/em\u003e analyzed, as the MIB-MIP pairs of the type-strain G5847\u003csup\u003eT\u003c/sup\u003e are similar to the ones present in \u003cem\u003eMccp\u003c/em\u003e, while the MIB-MIP pairs of the IVB14/OD_0535 strain cluster closely to the ones of the other members of the \u0026lsquo;\u003cem\u003eM. mycoides\u003c/em\u003e cluster\u0026rsquo;. Remarkably, the two paired MIB-MIP copies of \u003cem\u003eM. hyopneumoniae\u003c/em\u003e, located on different chromosomal loci, are highly divergent, with one MIB-MIP being more similar to the only system present in \u003cem\u003eM. hyorhinis\u003c/em\u003e and the other more similar to MIB-MIP present in \u003cem\u003eMesomycoplasma ovipneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Overall, our analysis shows how widespread these tandem systems are within the \u003cem\u003eMollicutes\u003c/em\u003e, with high sequence divergence and multiple possible gene acquisitions, particularly between species sharing the same hosts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of MIB-MIP expression in\u003c/b\u003e \u003cb\u003eM. feriruminatoris\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe studied the expression of each MIB-MIP gene pair of this species by transcriptomics and proteomics analyses to see whether the gene pairs are organized as operons and to identify different promotors that can be used to express MIB-MIPs from other \u003cem\u003eMollicutes\u003c/em\u003e species. Exploration of the transcriptomic data showed that each MIB-MIP pair constitutes an individual transcriptional unit, with the first and last pairs (MM1\u003csub\u003emfe\u003c/sub\u003e and MM4\u003csub\u003emfe\u003c/sub\u003e) being expressed at higher levels than the other two pairs (MM2\u003csub\u003emfe\u003c/sub\u003e and MM3\u003csub\u003emfe\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, all MIB-MIP gene pairs are among the mid-to-high expressed genes of \u003cem\u003eMferi\u003c/em\u003e, with no apparent differences between MIB and MIP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, when analyzing the proteomics data, MIB-MIP pairs are not among the highly expressed proteins of the cell highlighting the lack of correlation between RNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Besides, the expression levels of all MIP copies of each pair are significantly higher expressed compared to their MIB counterparts. Given the similar disposition and number of the different MIB-MIP gene pairs between \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 and \u003cem\u003eMmc\u003c/em\u003e GM12, we also analyzed transcriptomics and proteomics data of GM12 obtained in a previous study \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Our results showed a similar mRNA expression trend for each MIB-MIP pair, with the first tandem of genes being expressed higher than the rest (Supp. Fig. S2). Moreover, proteomics data showed that most MIP proteins are expressed at higher levels than their respective MIB counterparts, with most of them being not reliably detected (Supp. Fig. S2). Overall, these results suggest that each MIB-MIP system constitutes an individual expression unit and that expression of the different tandem of genes is species-specific, despite having similar genetic organization or distribution in the different chromosomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of MIB-MIP promoter and terminator regions\u003c/h2\u003e \u003cp\u003eTo further characterize the transcriptional profile shown by the RNAseq analysis of the MIB-MIP locus of \u003cem\u003eMferi\u003c/em\u003e, we decided to experimentally determine the transcriptional start sites (TSSs) present in that genomic area by primer extension. We could only reliably detect the TSS of the last MIB-MIP gene tandem encoded in \u003cem\u003eMferi\u003c/em\u003e, while no clear signal could be seen for the other gene pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The determination of the TSS of the elongation factor Tu gene (\u003cem\u003etufB\u003c/em\u003e) was used to establish the experimental conditions. Moreover, genetic analysis of the downstream regions of MIB-MIP gene clusters in a number of members of the '\u003cem\u003eMycoplasma mycoides\u003c/em\u003e cluster' revealed the presence of inverted repeat elements with a structure reminiscent of rho-independent terminators (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These elements are 32-34bp long and are located very close to the stop codon of the MIP genes. The last MIB-MIP gene pair is always devoid of this downstream element in all studied species, suggesting that it is part of a larger operon unit that also comprises putative ATPase-coding genes located downstream of the MIB-MIP cluster. The consensus sequence of these elements was determined as 5\u0026rsquo;TA(A/C)NATCCTTT(A/G)G-NT(A/T\u003csub\u003e2\u003c/sub\u003e)T(A/T\u003csub\u003e2\u003c/sub\u003e)-CTAAAGGATTTTT using all available sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Employing RNAfold \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, the RNA structure of these downstream elements was predicted to be a small hairpin with a minimum free energy of approximately \u0026minus;\u0026thinsp;12.70 kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration and characterization of a\u003c/b\u003e \u003cb\u003eM. feriruminatoris\u003c/b\u003e \u003cb\u003eΔMIB-MIP strain\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the effectiveness and versatility of the new \u003cem\u003eoriC\u003c/em\u003e-plasmids developed for \u003cem\u003eMferi\u003c/em\u003e we decided to test expression of the recently identified MIB-MIP cleavage systems. \u003cem\u003eMferi\u003c/em\u003e strain IVB14/OD_0535 has several genes coding for a total of four MIB-MIP tandem systems clustered in a single chromosomal locus \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, in a similar disposition as in \u003cem\u003eMmc\u003c/em\u003e GM12 \u003csup\u003e5\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To assess activity of each unique MIB-MIP gene pair of \u003cem\u003eMferi\u003c/em\u003e using oriC-plasmids \u003cem\u003ein cellulo\u003c/em\u003e, we aimed at the generation of a ΔMIB-MIP knock-out mutant by cloning and modifying the genome of \u003cem\u003eMferi\u003c/em\u003e in yeast prior transplantation of the modified genome to a new mycoplasma cell. The genome of \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 was transformed into \u003cem\u003eS. cerevisiae\u003c/em\u003e carrying the necessary plasmids to replace the chromosomal locus coding for the four MIB-MIP gene pairs (MF5583_00301 to MF5583_00308, ~\u0026thinsp;20Kb) by a recombination template using the CReasPy-Cloning method \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. YACs containing the modified genome were transplanted into an \u003cem\u003eMcap\u003c/em\u003e ΔRE recipient cell to obtain the desired \u003cem\u003eMferi\u003c/em\u003e ΔMIB-MIP strain (Figure S3). This knock-out mutant could grow similarly to the WT strain in the absence of antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), with a doubling time of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;2min compared to 45\u0026thinsp;\u0026plusmn;\u0026thinsp;3min of the WT in SP-5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Besides, the ΔMIB-MIP phenotype was confirmed by an IgG cleavage assay in which this mutant lost the ability to cleave the heavy chain of purified goat IgGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lane 4), compared to the wild-type strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lane 3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of native MIB-MIP gene pairs of\u003c/b\u003e \u003cb\u003eM. feriruminatoris in trans\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach unique MIB-MIP gene pair of \u003cem\u003eMferi\u003c/em\u003e was cloned in a pIVB09 backbone under the control of their own natural promoters previously determined by RNAseq or primer extension. The newly constructed \u003cem\u003eMferi\u003c/em\u003e ΔMIB-MIP strain was transformed with each of these plasmids individually. Positive clones were exposed to goat IgGs to assess cleavage activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Despite analyzing multiple clones harboring each MIB-MIP gene pair (data not shown), we could only detect IgG heavy chain cleavage in clones expressing the first and last gene tandems of the cluster (MM1\u003csub\u003emfe\u003c/sub\u003e and MM4\u003csub\u003emfe\u003c/sub\u003e), while other clones expressing the other MIB-MIP pairs (MM2\u003csub\u003emfe\u003c/sub\u003e and MM3\u003csub\u003emfe\u003c/sub\u003e) showed marginal cleavage or activity below the detection limit. As the transcriptomics data suggested that the MM1\u003csub\u003emfe\u003c/sub\u003e and MM4\u003csub\u003emfe\u003c/sub\u003e gene tandems were expressed at higher levels than MM2\u003csub\u003emfe\u003c/sub\u003e and MM3\u003csub\u003emfe\u003c/sub\u003e, and that expression from the promoter region of MM1\u003csub\u003emfe\u003c/sub\u003e (P\u003csub\u003eMM1mfe\u003c/sub\u003e) had no apparent interplay with any rho-independent terminator or upstream regulatory sequences, we decided to reintroduce the MM2\u003csub\u003emfe\u003c/sub\u003e and MM3\u003csub\u003emfe\u003c/sub\u003e gene pairs under the control of P\u003csub\u003eMM1mfe\u003c/sub\u003e in a ΔMIB-MIP genetic background. Under these conditions, all MIB-MIP gene pairs could be expressed, and mutants showed clear IgG cleavage activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), suggesting that all MIB-MIP gene pairs are functional \u003cem\u003ein cellulo\u003c/em\u003e, and that the P\u003csub\u003eMM1mfe\u003c/sub\u003e region was sufficient to drive expression of two relatively large membrane-associated proteins organized in an operon.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eIgG cleavage by\u003c/b\u003e \u003cb\u003eM. hyopneumoniae\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eM. hyorhinis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCleavage of immunoglobulins by the MIB-MIP system has been reported in \u003cem\u003eMollicutes\u003c/em\u003e of the formerly known '\u003cem\u003eSpiroplasma\u003c/em\u003e phylogenetic group', i.e \u003cem\u003eMmc\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003eMferi\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, but never in \u003cem\u003eMollicutes\u003c/em\u003e species like \u003cem\u003eMesomycoplasma\u003c/em\u003e spp. To determine if important porcine pathogens such as \u003cem\u003eM. hyopneumoniae\u003c/em\u003e or \u003cem\u003eM. hyorhinis\u003c/em\u003e can target and cleave host IgGs, we analyzed immunoglobulin cleavage activity of two strains isolated in Switzerland. Genome analysis revealed that \u003cem\u003eM. hyopneumoniae\u003c/em\u003e Ue273 contains two complete MIB-MIP gene pairs and a single orphan MIB gene, while \u003cem\u003eM. hyorhinis\u003c/em\u003e JF5820 only contains a single MIB-MIP gene pair \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This contrasts with many \u003cem\u003eMollicutes\u003c/em\u003e species of the \u0026lsquo;Spiroplasma phylogenetic group\u0026rsquo;, where all MIB-MIP gene copies are clustered in a single chromosomal locus containing 3\u0026ndash;4 complete MIB-MIP gene pairs. Interestingly, neither of the different MIB-MIP copies in \u003cem\u003eM. hyopneumoniae\u003c/em\u003e were downstream followed by an ATPase gene cluster, as it is the case in most \u003cem\u003eMollicutes\u003c/em\u003e species. This ATPase gene cluster in \u003cem\u003eM. hyopneumoniae\u003c/em\u003e is found in a different chromosomal location instead, seemingly controlled by a DNA slippage mechanism in a similar fashion as antigenic or phase variation switches present in \u003cem\u003eMollicutes\u003c/em\u003e (Figure S4) (Citti 2010). Incubation with purified commercial IgGs isolated from na\u0026iuml;ve pig serum showed cleavage activity by both pathogens \u003cem\u003eM. hyorhinis\u003c/em\u003e and \u003cem\u003eM. hyopneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), with the heavy chain of the immunoglobulins being targeted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous expression of MIB-MIP gene pairs of other\u003c/b\u003e \u003cb\u003eMollicutes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe availability of a ΔMIB-MIP strain together with a vector capable of expression of MIB-MIP pairs \u003cem\u003ein trans\u003c/em\u003e in \u003cem\u003eMferi\u003c/em\u003e prompted us to complement this strain with MIB-MIP systems from other \u003cem\u003eMollicutes\u003c/em\u003e species. First, we cloned the last gene tandem of the MIB-MIP operon of \u003cem\u003eMmc\u003c/em\u003e GM12 (MM4\u003csub\u003eMmc\u003c/sub\u003e) under the control of the promoter region of the first gene tandem of the same species (P\u003csub\u003eMM1Mmc\u003c/sub\u003e), mimicking a similar disposition performed \u003cem\u003ein situ\u003c/em\u003e at the chromosomal location in a previous work \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This construction was transformed in the ΔMIB-MIP strain and positive clones exhibit restored capacity to cleave goat IgGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, lane 6). Hereafter, we cloned the two complete MIB-MIP gene pairs and the single MIB-MIP gene tandem from \u003cem\u003eM. hyopneumoniae\u003c/em\u003e Ue273 (MM1\u003csub\u003eMhp\u003c/sub\u003e and MM2\u003csub\u003eMhp\u003c/sub\u003e) and \u003cem\u003eM. hyorhinis\u003c/em\u003e JF5820 (MM\u003csub\u003eMhr\u003c/sub\u003e) in a pIVB09 backbone. In a first attempt, we introduced each MIB-MIP set under the control of their natural promoters, as previously done with the MIB-MIP genes of \u003cem\u003eMferi\u003c/em\u003e and \u003cem\u003eMmc\u003c/em\u003e. However, despite obtaining similar number of transformants carrying the different \u003cem\u003eoriC\u003c/em\u003e-plasmids, no IgG cleavage was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To facilitate recombinant expression, we adapted all the MIB-MIP coding sequences to the codon usage of \u003cem\u003eMmc\u003c/em\u003e GM12, the closest species of the \u0026lsquo;\u003cem\u003eMycoplasma mycoides\u003c/em\u003e cluster\u0026rsquo; to \u003cem\u003eMferi\u003c/em\u003e with an available characterized codon usage table (kazusa.or.jp), and replaced the natural promoters with the P\u003csub\u003eMM1Mfe\u003c/sub\u003e, which proved capable of generating mRNA of similar length as previously shown in this study. However, transformants carrying these new constructs could neither cleave goat nor porcine IgGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), suggesting that the system was not active or could not be correctly exported, folded or displayed at the membrane of \u003cem\u003eMferi\u003c/em\u003e cells.\u003c/p\u003e \u003cp\u003eTo further investigate this, we decided to clone in pIVB09 tagged-versions of the MIB-MIP systems of \u003cem\u003eMferi\u003c/em\u003e (MM4\u003csub\u003eMfe\u003c/sub\u003e), \u003cem\u003eMmc\u003c/em\u003e (MM4\u003csub\u003eMmc\u003c/sub\u003e) and the single MIB-MIP system of \u003cem\u003eM. hyorhinis\u003c/em\u003e (MM\u003csub\u003eMhr\u003c/sub\u003e) to track protein expression by immunoblotting. All MIB genes were fused with a C-terminal 6xHis tag, while their MIP counterparts were tagged with a C-terminal FLAG tag. Analysis of \u003cem\u003eMferi\u003c/em\u003e ΔMIB-MIP strains carrying these plasmids showed that neither of the proteins forming the MIB-MIB system of \u003cem\u003eM. hyrorhinis\u003c/em\u003e was expressed in these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which explained the lack of IgG cleavage showed previously. Sequence analysis of the MIB-MIP systems of \u003cem\u003eMferi\u003c/em\u003e, \u003cem\u003eMmc\u003c/em\u003e and \u003cem\u003eM. hyorhinis\u003c/em\u003e showed significant differences in the N-terminal residues (Supp. Fig. S5), which could prevent export of these proteins to the cell surface. To test this, we replaced the predicted signal peptides of the tagged MIB-MIP system of \u003cem\u003eM. hyorhinis\u003c/em\u003e with the ones present in the MIB-MIP pair 1 of \u003cem\u003eMferi\u003c/em\u003e. Strains carrying this construction could correctly express the protease component of the MM\u003csub\u003eMhr\u003c/sub\u003e, but not the binding protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGoats infected intranasally and transtracheally with\u003c/b\u003e \u003cb\u003eMycoplasma feriruminatoris\u003c/b\u003e \u003cb\u003edid not develop disease\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMferi\u003c/em\u003e is considered a promising candidate for the development of a vaccine chassis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003eMferi\u003c/em\u003e has only been isolated from wild \u003cem\u003ecaprinae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, data regarding its pathogenic potential in closely related domestic animals are absent. Therefore, we decided to assess the pathogenicity of the type-strain G5847\u003csup\u003eT\u003c/sup\u003e of \u003cem\u003eMferi\u003c/em\u003e as the representative member of the species. We used a challenge model established for the phylogenetically related species \u003cem\u003eMmc\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and modified for \u003cem\u003eMccp\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, which is robust and reproducible \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Positive control was the highly virulent \u003cem\u003eMccp\u003c/em\u003e ILRI181 \u003csup\u003e49\u003c/sup\u003e. Clinical evaluation was assessed daily and was carried out 10 days pre-infection up to 25 days post-infection (dpi). Goats infected with \u003cem\u003eMferi\u003c/em\u003e showed no clinical signs in contrast to animals infected with \u003cem\u003eMccp\u003c/em\u003e, which showed onset of clinical disease including elevated body temperature at 6\u0026ndash;8 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This was followed by high fever (\u0026gt;\u0026thinsp;40.5\u0026deg;C for all animals), associated with respiratory distress, coughing and wheezing (8\u0026ndash;10 dpi), less movement and reduced intake of food. All criteria considered, this clinical evaluation led to a severity grade of 3 at 10 dpi; consequently, the three animals infected with \u003cem\u003eMccp\u003c/em\u003e were euthanized (Supp. Fig S6). During the course of infection, we monitored the hematological parameters. All animals infected with either species did not show a clear difference in the hematological parameters compared to their baseline levels prior infection (Supp. Fig S6). Postmortem analysis did reveal CCPP-typical pathomorphological changes including the detection of \u003cem\u003eMccp\u003c/em\u003e, while the animals infected with \u003cem\u003eMferi\u003c/em\u003e did not have any lesions pointing towards \u003cem\u003eMferi\u003c/em\u003e-related disease and \u003cem\u003eMferi\u003c/em\u003e could not be isolated from the animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eMferi\u003c/em\u003e has so far only been isolated from wild ruminants such as Alpine ibex \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The ability to modify its genome using synthetic genomics tools \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, the absence of the cell wall, its glycosylation capacity and its favorable growth attributes that compare to model organisms such as \u003cem\u003eE. coli\u003c/em\u003e make it an appealing candidate for vaccine- and drug delivery especially with reference to the respiratory tract or cancer treatment \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In this work, we investigated the pathogenicity of \u003cem\u003eMferi\u003c/em\u003e in domestic goats using an infection model that has been successfully used for phylogenetically closely related mycoplasmas. \u003cem\u003eMollicutes\u003c/em\u003e are reported to have high species tropism this should be confirmed for \u003cem\u003eMferi\u003c/em\u003e in an \u003cem\u003ein vivo\u003c/em\u003e experiment. Our data do not point to pathogenicity in domestic goats and therefore this organism is unlikely to infect even phylogenetically more distant organisms, which is important for safety concerns. Only one infection route was tested, which is the main one in closely related bacteria of the \u0026lsquo;\u003cem\u003eM. mycoides\u003c/em\u003e cluster\u0026rsquo; \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Goats challenged with \u003cem\u003eMccp\u003c/em\u003e, as expected, reached endpoint criteria at 10 dpi and were euthanized. \u003cem\u003eMferi\u003c/em\u003e could not be isolated from animals challenged with the latter, while \u003cem\u003eMccp\u003c/em\u003e was isolated from pathomorphological lesion of the animals infected with it. The absence of \u003cem\u003eMferi\u003c/em\u003e from the post-mortem tissues investigated supports the fact that the animals cleared \u003cem\u003eMferi\u003c/em\u003e from the system.\u003c/p\u003e \u003cp\u003eSurface expression of heterologous antigens in \u003cem\u003eMferi\u003c/em\u003e is desirable for future antigen presentation in a live vaccine chassis. We aimed to investigate heterologous surface expression and focused our work on the MIB-MIP system. To get an idea of the sequence conservation of the MIB-MIP system in \u003cem\u003eMollicutes\u003c/em\u003e we analyzed the presence and genetic identity in different species. It has been shown that genes homologous to the MIB-MIP system of \u003cem\u003eMmc\u003c/em\u003e were present frequently as several repeated gene copies in most species of \u003cem\u003eMycoplasma\u003c/em\u003e, \u003cem\u003eMycoplasmopsis\u003c/em\u003e, \u003cem\u003eMetamycoplasma\u003c/em\u003e and \u003cem\u003eMesomycoplasma\u003c/em\u003e, and a token presence in \u003cem\u003eMycoplasmoides\u003c/em\u003e spp. \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Here, we show that these systems are highly divergent in the different \u003cem\u003eMollicutes\u003c/em\u003e, with several copies being potentially exchanged between distant species sharing the same host. Horizontal transfer in these bacteria has been mainly attributed to the presence and activity of integrative-conjugative elements (ICE), first described in \u003cem\u003eMycoplasmopsis fermentans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and later studied in depth in \u003cem\u003eMycoplasmopsis agalactiae\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eMycoplasmopsis bovis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Some species of \u003cem\u003eMollicutes\u003c/em\u003e carry MIB-MIP tandem genes in close vicinity of ICE sequences, like the case of \u003cem\u003eMmc\u003c/em\u003e GM12 \u003csup\u003e8\u003c/sup\u003e, which could explain the dissemination of these immunoglobulin cleavage systems between species inhabiting the same niche. No traces of vestigial ICE could be found near the MIB-MIP sequences of \u003cem\u003eM. synoviae\u003c/em\u003e, \u003cem\u003eM. pulmonis\u003c/em\u003e, \u003cem\u003eM. arthritidis\u003c/em\u003e or \u003cem\u003eM. gallisepticum\u003c/em\u003e, however other ICE-independent conjugation mechanisms seem to exist among the \u003cem\u003eMollicutes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and could be used to transfer specific genomic sequences between different species. Another possibility could be the transfer of this genetic material via phage infection, as some species like \u003cem\u003eM. pulmonis\u003c/em\u003e or \u003cem\u003eM. arthritidis\u003c/em\u003e are susceptible to phage attacks \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, although genetic exchange via viral transduction has never been reported in \u003cem\u003eMollicutes\u003c/em\u003e to the best of our knowledge.\u003c/p\u003e \u003cp\u003eNext, we developed transformation protocols and oriC-type plasmid vectors to shuttle antigen-encoding genes into \u003cem\u003eMferi\u003c/em\u003e and to accelerate the testing of heterologous protein expression. Our growth curves of the wild-type strain of \u003cem\u003eMferi\u003c/em\u003e showed the rapid decline of viable cells after 20h of cultivation in SP-5 medium, coinciding with medium acidification below pH7. This characteristic contrasts slightly \u003cem\u003eMmc\u003c/em\u003e, a closely related relatively fast-growing \u003cem\u003eMycoplasma\u003c/em\u003e species, which can survive and maintain high bacterial titers for longer time in acidic conditions. Loss of viability upon acidification in mycoplasmas is not exceptional \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, thus the differences in low pH tolerance between \u003cem\u003eMferi\u003c/em\u003e and \u003cem\u003eMmc\u003c/em\u003e could be attributed to distinctive metabolic capabilities \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e or growth requirements in both species. This reduced tolerance towards lower pH prompted us to adapt the standard transformation protocols for mycoplasmas of the '\u003cem\u003eM. mycoides\u003c/em\u003e cluster', which use cells harvested at pH 6.2\u0026ndash;6.5 and PEG solutions buffered at a similar pH, for transformation of \u003cem\u003eMferi\u003c/em\u003e. Most transformable \u003cem\u003eMollicutes\u003c/em\u003e species have higher transformation efficiencies when harvested at late-log phase \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. By increasing the initial pH of the SP-5 medium from 7.5 to 8, we could significantly increase the bacterial titers of \u003cem\u003eMferi\u003c/em\u003e after an overnight growth to 1-3x10\u003csup\u003e9\u003c/sup\u003e CFU/mL at pH 7, right before cell titer decline, optimizing transformation efficiencies for this bacterium. Thus, we also adapted the pH of all transformation solutions to pH 7 to mimic the medium conditions at the harvesting point.\u003c/p\u003e \u003cp\u003eIn this work we developed a series of replicative plasmids based on the modification of the origin of replication of the type-strain G5847\u003csup\u003eT\u003c/sup\u003e that can be easily used in \u003cem\u003eMferi\u003c/em\u003e. Many species of \u003cem\u003eMollicutes\u003c/em\u003e can stably maintain episomal DNA containing the \u003cem\u003eoriC\u003c/em\u003e sequence of the same species or a closely related one \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. For certain species such as \u003cem\u003eM. agalactiae\u003c/em\u003e, it has been found that the \u003cem\u003ednaA\u003c/em\u003e gene present in these \u003cem\u003eoriC\u003c/em\u003e plasmids is not essential for replication and propagation. The removal of \u003cem\u003ednaA\u003c/em\u003e and simplification of the \u003cem\u003eoriC\u003c/em\u003e region results in less frequent integration events at the chromosomal \u003cem\u003eoriC\u003c/em\u003e locus in most species \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, with the cost of usually lower transformation efficiency rates. On the contrary, in the case of \u003cem\u003eoriC\u003c/em\u003e-plasmids derived from \u003cem\u003eMferi\u003c/em\u003e, removal of the \u003cem\u003ednaA\u003c/em\u003e gene resulted in 5 times higher transformation efficiencies regardless of the antibiotic marker used. Only one \u003cem\u003eoriC\u003c/em\u003e-plasmid developed in this work, pIVB04, in which the \u003cem\u003etetM\u003c/em\u003e marker had been replaced with \u003cem\u003epac\u003c/em\u003e marker did not yield any transformants despite having the same configuration as pIVB03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was only when the orientation of the \u003cem\u003epac\u003c/em\u003e marker was flipped (plasmid pIVB06) that the plasmid yielded a similar number of transformants than pIVB03. This fact suggests that the \u003cem\u003eoriC\u003c/em\u003e region contains promoter sequences that could challenge the transcription of the pS'\u003cem\u003epac\u003c/em\u003e cassette by antisense inhibition of gene expression. Antisense RNA-mediated transcriptional attenuation in bacteria is well described \u003csup\u003e\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, involving either dsRNA-specific RNases, peptide nucleic acids, phosphorodiamidate morpholino oligomers or just by steric hindrance of transcription or translation. In \u003cem\u003eMollicutes\u003c/em\u003e, antisense RNAs have been identified in pathogenic species of swine such as \u003cem\u003eM. hyopneumoniae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and human species like \u003cem\u003eM. pneumoniae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003eM. genitalium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and their role in modulation of gene expression has been acknowledged. However, it seems likely that by steric hindrance, the RNA polymerase complex cannot successfully read through two genes with colliding orientations if both promoters are spatially close, which should result in a lower expression of both transcripts. This was shown in \u003cem\u003eM. genitalium\u003c/em\u003e, when transposons expressing the toxic MG_428 gene coding for the alternative sigma factor σ\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e were all inserted in highly expressed genes in the opposite orientation of transcription, which dampened expression of the toxic gene and allowed cells to live \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the case of pIVB04, most likely the promoter of the \u003cem\u003ednaN\u003c/em\u003e gene is interfering with the expression of the \u003cem\u003epac\u003c/em\u003e marker controlled by the spiralin promoter pS', interfering with the expression of the antibiotic cassette and limiting the puromycin resistance of transformed cells with the \u003cem\u003eoriC\u003c/em\u003e-plasmid. This is not the case for pIVB03 and pMYCO1 \u003cem\u003eoriC\u003c/em\u003e-plasmids, likely due to the larger size of the \u003cem\u003etetM\u003c/em\u003e marker (1.9kb) compared to the \u003cem\u003epac\u003c/em\u003e cassette (0.6kb).\u003c/p\u003e \u003cp\u003eAccording to the \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535 genomic sequence, this strain carries four MIB-MIP copies clustered together in the same genetic locus. Our transcriptomics analyses showed that each pair is an individual transcriptional unit - an operon - with its own putative promoter and a short palindromic sequence resembling rho-independent terminator sequences. We showed that the promoter of the first and last gene tandems are significantly stronger than the other two, which may influence the transcription of downstream elements. The first promoter element may overcome the terminator element and drive the expression of other MIB-MIP pairs, while the last promoter likely plays a role on the expression of the highly conserved putative ATP-synthase gene cluster situated immediately downstream. It is still not clear if MIB-MIP-related IgG cleavage requires the activity of the downstream ATPase, but our results show that the two gene clusters do not necessarily work \u003cem\u003ein cis\u003c/em\u003e. In keeping with this, the MIB-MIP systems present in \u003cem\u003eM. hyopneumoniae\u003c/em\u003e are unlinked to the ATPase gene cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Supp. Figure S4), which is still present but in a different genetic context in this species. Due to their reduced-size genomes, \u003cem\u003eMollicutes\u003c/em\u003e are thought to be devoid of many transcriptional regulatory elements, and rho-independent terminators have been suggested as major fine-tuning, transcription-controlling elements \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, in conjunction with DNA supercoiling and RNA degradation \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. We could demonstrate that the promoter elements of the first MIB-MIP gene copy of \u003cem\u003eMferi\u003c/em\u003e (P\u003csub\u003eMM1mfe\u003c/sub\u003e) was capable of successfully drive the expression of all MIB-MIP gene tandems individually \u003cem\u003ein trans\u003c/em\u003e, which makes it a promising tool for recombinant expression of other rather large membrane proteins using this bacterium in the future.\u003c/p\u003e \u003cp\u003eDespite several attempts, expression of active heterologous MIB-MIP systems could only be achieved with a MIB-MIP system of \u003cem\u003eMmc\u003c/em\u003e, which is phylogenetically closely related to \u003cem\u003eMferi\u003c/em\u003e. Expression of functional MIB-MIP gene tandems from the porcine \u003cem\u003eMollicutes\u003c/em\u003e species \u003cem\u003eM. hyopneumoniae\u003c/em\u003e and \u003cem\u003eM. hyorhinis\u003c/em\u003e was not possible, despite the use of native promoters from \u003cem\u003eMferi\u003c/em\u003e or adapting the codon usage. The structure of the MIB-MIP tandem system has been recently obtained and characterized \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and shows direct contacts between the two protein counterparts and with the targeted immunoglobulin. Therefore, correct export and folding of both proteins is likely pivotal for the system to work. We show that IgG cleavage of \u003cem\u003eM. hyopneumoniae\u003c/em\u003e and \u003cem\u003eM. hyorhinis in cellulo\u003c/em\u003e is possible under the standard laboratory conditions, which indicates that the systems present in these bacteria are active when expressed correctly. Despite our efforts, we failed at expressing active MIB-MIP systems from distantly related \u003cem\u003eMollicutes\u003c/em\u003e in \u003cem\u003eMferi\u003c/em\u003e, most likely due to problems related to the export of the system to the membrane. Despite many years of research, the protein export systems of \u003cem\u003eMollicutes\u003c/em\u003e are poorly characterized\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Most MIP proteins analyzed in this work contain a classic lipoprotein signal peptide (type II signal peptide), which consist of a positively charged N-terminal region, followed by a central hydrophobic area and a polar C-terminal region (lipobox) \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. This signal peptide is also known to contain a preserved motif LXXC, which is recognized by the preprolipoprotein diacylglyceryl transferase (Lgt, encoded in MF5583_00077 and 00079 in \u003cem\u003eMferi\u003c/em\u003e IVB14/OD_0535) followed by the apolipoprotein \u003cem\u003eN-\u003c/em\u003eacyltransferase (Lnt, encoded in MF5583_00341) that will create the linkage of the protein to the cell membrane after translocation via the Sec pathway \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. However, none of the MIB proteins analyzed have a similar type II signal peptide or any clear transmembrane domain that suggests \u003cem\u003ein silico\u003c/em\u003e association at the cell surface (Supp. Fig. S7), aside from the interaction with MIP required for immunoglobulin cleavage. Furthermore, very low MIB protein levels are detected in our proteomics analyses in either \u003cem\u003eMmc\u003c/em\u003e or \u003cem\u003eMferi\u003c/em\u003e, as it was previously reported in \u003cem\u003eMmc\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition, in another closely related \u003cem\u003eMycoplasma\u003c/em\u003e species namely \u003cem\u003eMmm\u003c/em\u003e, only the MIP proteins have been clearly detected in the surface proteome \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Similarly, the closely related protein M, present in other \u003cem\u003eMollicutes\u003c/em\u003e species usually devoid of MIB-MIP systems \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, also lacks any clear membrane anchoring signal and could not be identified in the protein membrane enriched fractions or cell-surface protein labelling in a thorough proteomics study carried out in \u003cem\u003eM. genitalium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. However, a recent study characterizing the protein M homolog (IbpM) from \u003cem\u003eMycoplasmoides pneumoniae\u003c/em\u003e shows data indicating that this protein is located at the cell surface \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e, despite that advanced transmembrane domain predictors like DeepTMHMM \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e do not predict the presence of any transmembrane domain in neither protein M nor MIB. Understanding how these and other proteins lacking conventional signal peptides are exported in \u003cem\u003eMferi\u003c/em\u003e is crucial to develop functional display systems in this bacterium.\u003c/p\u003e \u003cp\u003eIn conclusion, in this work we assessed pathogenicity of \u003cem\u003eMferi\u003c/em\u003e in an established animal model, developed new oriC-based vectors for rapid and versatile gene delivery in this microorganism and use them to characterize expression of native and foreign anti-immunoglobulin systems of \u003cem\u003eMollicutes\u003c/em\u003e, providing new data regarding the molecular mechanisms of these specialized machineries that should aid in the understanding of the immune evasion strategies of pathogenic \u003cem\u003eMollicutes\u003c/em\u003e species. Moreover, we identified promotors suitable to drive expression of rather large heterologous surface proteins, which will be pivotal for future applications of \u003cem\u003eMferi\u003c/em\u003e as a bacterial vaccine chassis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eJJ and ST-P conceived the project and planned the experiments. ST-P, SC-P, FL and TY carried out the laboratory experimental work. ST-P and VC prepared the samples for -omics analyses. HA and PK performed the bioinformatics analyses. HP, NR, JJ and TD carried out the animal experimentation or analyzed the clinical data. ST-P and JJ drafted the manuscript. All authors read and agreed to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the Swiss National Science Foundation (grant number 310030_201152, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://benchling.com\" target=\"_blank\"\u003ewww.snf.ch\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.snf.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003ein vivo\u003c/em\u003e experiment was supported by the International Development Research Centre (Grant ID:108625). We thank the Lausanne Genomic Technologies Facility (University of Lausanne) for genome sequencing of the strains used in this work. We thank the Core Facility Proteomics \u0026amp; Mass Spectrometry (University of Bern) for their assistance in the proteomics analyses. We are grateful to the staff of the Institute for Virology and Immunology (IVI) for their assistance with the animal experimentation. We thank Isabelle Brodard and Bettina Tr\u0026uuml;eb for their assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNamba, S.: Molecular and biological properties of phytoplasmas. \u003cem\u003eProceedings of the Japan Academy Series B: Physical and Biological Sciences\u003c/em\u003e vol. 95 Preprint at (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2183/pjab.95.028\u003c/span\u003e\u003cspan address=\"10.2183/pjab.95.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer, A., et al.: The origin of the \u0026lsquo;\u003cem\u003eMycoplasma mycoides\u003c/em\u003e cluster\u0026rsquo; coincides with domestication of ruminants. 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(2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2022.04.08.487609\u003c/span\u003e\u003cspan address=\"10.1101/2022.04.08.487609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mycoplasma feriruminatoris, genome transplantation, oriC-plasmids, immunoglobulin cleavage, pathogenic porcine Mollicutes","lastPublishedDoi":"10.21203/rs.3.rs-3854399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3854399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate virulence factor present in multiple pathogenic species of the \u003cem\u003eMollicutes\u003c/em\u003e, including the fast-growing species \u003cem\u003eMycoplasma feriruminatoris\u003c/em\u003e. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work we analyzed the distribution and genetic relatedness between MIB-MIP systems of different \u003cem\u003eMollicutes\u003c/em\u003e species. Using -omics technologies, we show that the four copies of the \u003cem\u003eM. feriruminatoris\u003c/em\u003e MIB-MIP system have different expression levels, are transcribed as operons controlled by four different promotors. Individual MIB-MIP gene pairs of \u003cem\u003eM. feriruminatoris\u003c/em\u003e and other \u003cem\u003eMollicutes\u003c/em\u003e were introduced in an engineered \u003cem\u003eM. feriruminatoris\u003c/em\u003e strain devoid of MIB-MIP genes and were tested for their functionality using \u003cem\u003eoriC\u003c/em\u003e-based plasmids. The two proteins were functionally expressed at the surface of \u003cem\u003eM. feriruminatoris\u003c/em\u003e, which confirms the possibility to display large functional heterologous surface proteins in \u003cem\u003eM. ferirumintoris\u003c/em\u003e. Functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine \u003cem\u003eMollicutes\u003c/em\u003e like \u003cem\u003eMesomycoplasma hyorhinis\u003c/em\u003e or \u003cem\u003eMesomycoplasma hyopneumoniae\u003c/em\u003e could not be achieved. Finally, since \u003cem\u003eM. feriruminatoris\u003c/em\u003e is a candidate for biomedical applications such as drug delivery, we confirmed its safety \u003cem\u003ein vivo\u003c/em\u003e in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.\u003c/p\u003e","manuscriptTitle":"Characterization of the MIB-MIP system of different Mollicutes using an engineered Mycoplasma feriruminatoris","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 18:23:35","doi":"10.21203/rs.3.rs-3854399/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0664eacc-10dc-4ced-9e61-d3d3f708aaa6","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28179080,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":28179081,"name":"Biological sciences/Microbiology/Microbial genetics/Bacterial genes"}],"tags":[],"updatedAt":"2024-06-29T07:07:28+00:00","versionOfRecord":{"articleIdentity":"rs-3854399","link":"https://doi.org/10.1038/s42003-024-06497-8","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2024-06-28 04:00:00","publishedOnDateReadable":"June 28th, 2024"},"versionCreatedAt":"2024-01-30 18:23:35","video":"","vorDoi":"10.1038/s42003-024-06497-8","vorDoiUrl":"https://doi.org/10.1038/s42003-024-06497-8","workflowStages":[]},"version":"v1","identity":"rs-3854399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3854399","identity":"rs-3854399","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}