Antimicrobial activity screening of Bacteroidota and genome-based analysis of their antimicrobial biosynthetic potential

Antimicrobial activity screening of Bacteroidota and genome-based analysis of their antimicrobial biosynthetic potential | 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 Research Article Antimicrobial activity screening of Bacteroidota and genome-based analysis of their antimicrobial biosynthetic potential Diego Garcia-Morena, Maria Victoria Fernandez-Cantos, Willem Maathuis, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3875369/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The oral and gut microbiota constitute vastly diverse and complex ecosystems. Their presence affects local and distal organs, thus having a major role in health and disease. Bacteria forming these complex communities display social behaviour and can positively or negatively impact their neighbours. While the potential for antimicrobial production of Gram-positive bacteria has been widely investigated, the research on Gram-negative bacteria is lagging behind, also because current bioinformatic tools appear to be suboptimal to detect antimicrobial clusters in these bacteria. The present study investigates the antimicrobial potential of the Gram-negative Bacteroidota phylum members from oral and gut bacterial microbiota. For this purpose, several Bacteroidota strains of oral and gut origin were tested against each other, and the genomes of bacterial strains displaying interesting antimicrobial activity were mined. Several biosynthetic gene clusters were detected, and the potential peptide sequences were identified. These putative peptides showed low sequence similarity to each other. Still, all contained a Gly-Gly motif, probably representing the processing site of the prepeptide, and they shared a similar N-terminal region reminiscent of the TIGR04149 protein family. However, the cluster architecture differed between the biosynthetic gene clusters, indicating they contain different posttranslational modifications (PTMs). These findings highlight the potential for novel antimicrobial discovery in Gram-negative bacteria relevant to the human microbiota and their ecology. General Microbiology Bioinformatics Oral microbiota gut microbiota antimicrobial activity Bacteroidota genome mining Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Along the gastrointestinal (GI) tract, the presence of (semi)processed nutrients creates an ideal environment for a wide variety of microorganisms to thrive, with some of the most dense bacterial communities found in the human oral cavity and the colon.[1] In both the oral cavity and the intestine, the predominant phyla of bacteria are the Firmicutes and the Bacteroidetes (Bacillota and Bacteroidota, hereafter)[2]. However, the balance between them changes depending on the location in the GI tract.[3–7] It has been shown that bacteria play a vital role in the metabolism and bioavailability of carbohydrates, amino acids, and certain vitamins and the correct development of the immune system.[8] Moreover, the presence of beneficial and neutral bacteria along the GI tract can prevent the establishment of harmful bacteria by outcompeting potential pathogens and excretion of bacterial antimicrobial compounds.[9] However, bacteria in the gut microbiota can also cause various diseases by disturbing the epithelial lining, causing cell death and creating an entrance for bacteria into the bloodstream, affecting proximal and distal organs. Recent research has shown that even without direct signs of cell death, modifications in the microbiota composition are associated with many diseases, such as irritable bowel syndrome (IBS), diabetes, cardiovascular disease and Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as Non-Alcoholic Fatty Liver Disease (NAFLD).[10–15] However, the bacterial gut microbiota is not the sole driver of disease. The oral microbiota has a major role in oral health, and recent evidence links its impact on systemic health and disease (recently reviewed by Baker et al., 2023).[16] To control these large communities of microorganisms, the body maintains a multi-layered defence. The established GI microbiota is also a part of this multi-layered defence, partly controlling its own quantitative and qualitative composition. Bacteria in the GI tract excrete various bacterial antimicrobial compounds to gain a relative advantage over competing species or members of the same genus. Bacteriocins, in particular, are a family of natural products with antimicrobial activity, comprising several families, such as the ribosomally synthesised and post-translationally modified peptides (RiPPs). These peptides present a huge structural variability due to the vast array of post-translational modifications (PTM) generated by enzymes encoded by adjacent genes.[17–19] Radical-SAM (rSAM) enzymes have been widely reported to be involved in peptide modification, defining post-translational modifications on Epipeptides,[20] Streptide,[21] Mycofactocin,[22,23] Pyrroloquinoline quinone,[24] Ranthipeptides,[25] Ryptides,[26] Sactipeptides[27,28] and Spliceotides.[29] Within the vast landscape of rSAM proteins, an increasing number of enzymes contain the SPASM/twitch domain, initially termed after maturases of S ubtilosin, P yrroloquinoline quinone (PQQ), A naerobic S ulfatases, and M ycofactocin.[30] In this family, besides the common CX 3 CX 2 C [4Fe-4S] cluster binding motif in the N-terminal region, members of the SPASM/twitch domain also contain a conserved 7-Cys motif (CX 9 – 15 GX 4 C-gap-CX 2 CX 5 CX 3 C-gap-C) in the C-terminus.[31,32] This motif is involved in the binding of one (twitch) or two (SPASM) auxiliary [4Fe-4S] clusters.[33–35] The three conserved cysteine residues in the CX 3 CX 2 C motif bind one iron each from the [4Fe-4S] cluster, leaving the fourth iron as a ligand for S -adenosylmethionine (SAM), which facilitates the reductive cleavage of SAM rendering methionine and a 5′-deoxyadenosyl radical, which will be used in other enzymatic reactions.[31,35,36] Intra- and/or inter-specific antimicrobial activity could be used as a gut microbiota modulator to prevent the advent or progression of such aforementioned diseases. The present study explores the antimicrobial potential in strains of the Bacteroidota and Fusobacteriota phyla. To achieve this, antimicrobial assays were performed with gut- and oral-isolated Bacteroidota species. When interactions were found, antimicrobial BGCs were mined and analysed. This revealed potential new peptides in Bacteroides sp. 4_1_36, B. Fragilis 3_1_12 and in Prevotella melaninogenica D18 able to aid in targeted microbiome modulation. Material and methods Sample collection, bacterial strains and culture conditions Bacterial strains used in this study are listed in Table 1 , showing the names, shorthand code, source, and sampling origin (if known). Table 2 collects the bacterial pathogenic strains used in the ‘Pathogen interactions’ experiment. Strains were provided by BEI Resources and the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. Table 1 List of all anaerobic strains, showing the full scientific and strain name, the shorthand code used, the source and, if known, the donor's sample location and health status. Scientific name Code Source Origin, health Bacteroides fragilis 3_1_12 Bf1 BEI Colon, healthy Bacteroides fragilis CL03T12C07 Bf2 BEI Faeces, healthy Bacteroides fragilis CL05T00C42 Bf3 BEI Faeces, healthy Bacteroides fragilis CL07T00C01 Bf4 BEI Faeces, healthy Bacteroides fragilis CL07T12C05 Bf5 BEI Faeces, healthy Bacteroides salyersiae CL02T12C01 B. sal2 BEI Faeces, healthy Bacteroides sp. 4_1_36 B6 BEI Ileum, Crohn’s disease Bacteroides stercoris DSM 19555 B. ster1 DSMZ Faeces, unknown Bacteroides vulgatus DSM 1447 Bv2 DSMZ Faeces, unknown Bacteroides xylanisolvens DSM 18836 Bx2 DSMZ Faeces, unknown Fusobacterium gonidiaformans CMW8396 CMW BEI Bacterial vaginosis Fusobacterium nucleatum animalis D11 D11 BEI Colon, inactive Crohn’s disease Fusobacterium nucleatum animalis F0419 F0419 BEI Oral cavity, healthy Fusobacterium nucleatum CTI-01 CTI-01 BEI Colon, colonic carcinoma Fusobacterium nucleatum CTI-02 CTI-02 BEI Colon, colonic carcinoma Fusobacterium nucleatum CTI-03 CTI-03 BEI Colon, colonic carcinoma Fusobacterium nucleatum CTI-05 CTI-05 BEI Colon, colonic carcinoma Fusobacterium nucleatum CTI-06 CTI-06 BEI Colon, colonic carcinoma Fusobacterium nucleatum CTI-07 CTI-07 BEI Colon, colonic carcinoma Fusobacterium nucleatum MJR7757B MJR7757B BEI Vaginal swab, pregnant Fusobacterium nucleatum polymorphum F0401 F0401 BEI Trachea, healthy Fusobacterium sp. CM21 CM21 BEI Oral cavity, unknown Fusobacterium sp. CM22 CM22 BEI Oral cavity, unknown Fusobacterium sp. F0437 F0437 BEI Oral cavity, unknown Fusobacterium sp. OBRC1 OBRC1 BEI Oral cavity, unknown Fusobacterium ulcerans 12_1B 12_1b BEI Colon, Crohn’s disease Parabacteroides distasonis 31_2 Par1 BEI Faeces, unknown Parabacteroides goldsteinii CC87F Pargo1 BEI Colon, healthy Parabacteroides johnsonii CL02T12C29 Parjo1 BEI Faeces, healthy Parabacteroides merdae CL03T12C32 Parme1/P1 BEI Faeces, healthy Parabacteroides sp. D13 D13 BEI Colon, ulcerative colitis Phocaeicola dorei 5_1_36/D4 Bd3 BEI Ileum, Crohn’s Phocaeicola dorei CL02T00C15 Bd4 BEI Faeces, healthy Phocaeicola dorei CL02T12C06 Bd2 BEI Faeces, healthy Phocaeicola dorei CL03T12C01 Bd1 BEI Faeces, healthy Porphyromonas gingivalis F0185 F0185 BEI Oral cavity, periodontitis Porphyromonas sp. KLE1280 KLE1280 BEI Oral cavity, plaque Porphyromonas sp. W7784 W7784 BEI Oral cavity, plaque Prevotella buccae D17 D17 BEI Oral cavity, healthy Prevotella denticola F0289 F0289 BEI Oral cavity, unknown Prevotella histicola F0411 F0411 BEI Oral cavity, plaque Prevotella melaninogenica D18 D18 BEI Oral cavity, healthy Prevotella nigrescens CC14M CC14M BEI Colon, unknown Prevotella oralis CC98A CC98A BEI Colon, unknown Prevotella oralis HGA0225 HGA BEI Colonic mucosa, unknown Treponema denticola AL-2 TP1 BEI Oral cavity, periodontal pocket Treponema denticola ASLM/F0460 TP2 BEI Oral cavity, periodontal pocket Treponema denticola H1-T TP3 BEI Oral cavity, periodontal pocket Treponema denticola H22 TP4 BEI Oral cavity, periodontal pocket Treponema denticola OTK TP5 BEI Oral cavity, periodontal pocket Treponema denticola US-trep/F0459 TP6 BEI Oral cavity, periodontal pocket All anaerobic strains were grown at anaerobic conditions in a Coy Anaerobic chamber. The atmospheric conditions were set to 1.5-2.0% H 2 , 5% CO 2 and 90+% N 2 . Strains were grown at 37°C without shaking. Bacteroides , Parabacteroides , and Phocaeicola strains were eventually grown in Gifu Anaerobic Broth from HiMedia (GAM; EWC Diagnostics Trade BV) supplemented with 5 µg/mL hemin and 2.5 µg/mL vitamin K1. The supplemented GAM was named GAM complete, or GAM c hereafter. Fusobacterium , Porphyromonas , and Prevotella strains were grown in Tryptone Soya Broth (TSB; Boom B.V.) supplemented with 250 µg/mL hemin and 2.5 µg/mL vitamin K1. The supplemented TSB was named TSB hemin 50x, or TSB h50 hereafter. GAM c and TSB h50 agar plates were prepared by adding 1.5% agar to the respective broth before autoclaving. Soft agar plates were prepared by adding 0.7% agar to each respective broth before autoclaving. In all cases, supplementation was done after autoclaving. All liquid media were introduced in the anaerobic chamber overnight; agar plates were cast right after supplementation and placed inside the COY anaerobic chamber overnight. Soft agar, autoclaved just before use, was allowed to degasify for 2 min under 20mm Hg of vacuum before being introduced into the anaerobic chamber and then supplemented inside. Table 2 List of all aerobic strains used, showing the full scientific and strain name and the shorthand code used. Scientific name Code Pseudomonas aeruginosa WT Psa Klebsiella pneumoniae LMG 20218 Kp Salmonella enterica LMG 07233 Se Acinetobacter baumannii LMG 01041 Ab Escherichia coli LMG 15862 Ec Candida albicans WT Ca Enterobacter aerogenes LMG 02094 Ea Enterobacter cloacae LMG 02783 Ec Screening of antimicrobial activity Colony antimicrobial assays: To find antimicrobial interactions between Bacteroidota species, GAM c and TSB h50 agar plates were poured and spotted with 10–15 different strains each. The following day, soft (0.7%) agar of the corresponding base media was prepared. Each spotted plate was assigned one of the spotted strains for its overlay, consisting of 1 mL liquid culture mixed into 14 mL 0.7% agar. As shown in Fig. 1 , on the third day, the plates were checked for halos, indicating antimicrobial activity. Pathogen interactions: To study the potential role of Bacteroidota strains in preventing infections caused by common pathogenic bacterial strains, an interaction assay was performed on a 96-well plate. 8 pathogenic strains (Table 2 ) were chosen and grown overnight in 4 mL of LB media at 37°C and 220 rpm. The pathogenic strains were diluted to OD 600 = 0.0005 in fresh LB, and 100 µL was added to the corresponding wells in the 96-well plate. 5 Bacteroidota strains were chosen for this experiment, namely B6, Bd2, Bf6, D18, and Parme1. The supernatant of these strains was obtained twice by centrifugation for 3 min at 12,000 rpm. 10 µL of an overnight culture of the pathogenic strains was used to inoculate respective soft agar tubes. On top of an agar layer, 5 mm metal rings were placed, and the inoculated overlay was poured around these rings. After removing the rings, the wells formed were filled with 75 µL of the centrifuged supernatant. For the positive and negative control, 50 µL of fresh GAMc was added in place of Bacteroidota supernatant. The 96-well plate was incubated overnight at 37°C and visually checked the next day. Antimicrobial compounds mining and metabolite biosynthetic gene cluster analysis To identify potential candidate RiPP BGCs, genome assemblies of the candidate strains were obtained from NCBI and the bioinformatic tools BAGEL4[37] and AntiSMASH[38] were used. The identification process involved considering the presence and arrangement of PTM enzymes, particularly rSAM ORFs, putative precursor peptides, and transporters within the area of interest (AOI) from BAGEL4 or the identified clusters from AntiSMASH. The selected AOIs, usually comprising over 20kbs sequence including the detected features, were manually investigated using the InterPro web-based tool,[39] Domain Architecture Retrieval Tool (CDART) and protein–protein BLAST in the NCBI web service. For rSAM protein clustering and identification, as well as for Genome Neighbouring Analysis, https://radicalsam.org/ )[40] was used. Finally, the amino acid sequence of relevant enzymes was obtained using their UniProt IDs and aligned in Jalview 2.11.2.7[41] using MUSCLE[42] on default parameters. Jalview 2.11.2.7 was also used for multiple sequence alignment (MSA) visualisation and Logo generation. Results Modified TSB supports the growth of several bacterial strains from the oral microbiota. While GAM c was a suitable media for Bacteroides strains, overnight inocula of Fusobacterium, Porphyromonas, Prevotella and Treponema strains in GAM c resulted in undetectable growth ( Supplementary Table S1 ). To improve the growth conditions, TSB medium was selected, supplemented with 5 µg/mL hemin and 2.5 µg/mL vitamin K1. This enhanced the growth of several Fusobacterium species. However, Prevotella , Porphyromonas, and Treponema species could still not grow on this medium . Therefore, TSB supplemented with increased concentrations of hemin was tested, as hemin is an essential growth factor for many Bacteroidota species.[43,44] This proved successful for Prevotella and Porphyromonas species , which grew reliably in TSB supplemented with 500 µg/mL hemin and 2.5 µg/mL vitamin K1, but not for Treponema species ( Supplementary Table S1 ). In fact, Treponema was not able to grow in TSB h50 supplemented with L-cysteine, which can function as an oxygen scavenger, pulling oxygen out of the solution.[45,46] Antimicrobial interactions Antimicrobial tests were conducted in TSB h50 ( Table 3) and GAMc (Table 4) using bacterial microbiota strains as producers and sensitive strains. The colony antimicrobial assays revealed that in the conditions studied, Bacteroides sp. 4_1_36 (B6) showed antimicrobial activity against members of the Fusobacterium , Porphyromonas and Bacteroides genera but not against the tested Prevotella species ( Tables 3 & 4 ). On the other hand, under these conditions, Prevotella melaninogenica D18, originally isolated from an oral swab from a healthy male patient in Canada[47] inhibited the growth of 10 other strains, including members of Prevotella , Fusobacterium , Porphyromonas and Bacteroides ( Table 3 ). In particular, D18 showed antimicrobial activity against five other oral strains and four gut strains. Interestingly, two of those five gut strains were: B. stercoris DSM 19555 (Bster1) and B. salyersiae CL02T12C01 (Bsal1). Finally, B. fragilis 3_1_12 (Bf1) showed intra-species antimicrobial activity,[48] inhibiting the growth of other B. fragilis strains, while unable to inhibit the growth of other members of the same genus, except for B. stercoris DSM 19555 ( Table 4 ). The antimicrobial activity found in B6, D18 and Bf1 against B. stercoris DSM 19555 and B. salyersiae CL02T12C01 is very relevant since these species had been correlated to the progress of MAFLD.[49] Therefore, finding bacterial strains able to impair their growth might prove helpful in developing new ways of dealing with MAFLD. Following the results from the antimicrobial assays, several strains were selected to further study their influence against known pathogens. However, the antimicrobial test showed no inhibitory effect ( Supplementary Table S2 ). The antimicrobial screenings ( Tables 3 & 4 ) revealed P. melaninogenica D18 as a strain with broad-spectrum antimicrobial activity, and both B. fragilis 3_1_12 (Bf1) and Bacteroides sp. 4_1_36 (B6) as strains with a narrower antimicrobial spectrum. Moreover, all three strains showed activity against B. stercoris DSM 19555, a strain related to MAFLD progression.[49] Therefore, these strains were selected for antimicrobial cluster mining to identify the putative biosynthetic gene cluster (BGC) involved in the halo formation. Biosynthetic gene cluster analysis reveals two novel putative antimicrobial clusters in Bacteroides sp . 4_1_36 Genome mining of B6 with BAGEL4 revealed one BGC of interest ( Figure 2A ), termed BGC1 hereafter. BGC1 encodes 17 ORFs, including one sequence predicted to be rSAM-modified_RiPP_057 (A1), which belongs to the TIGR04149 protein family or peptides associated with peptide-modifying radical SAM enzymes. Peptides in this family present a characteristic modular sequence, including a leader sequence with a conserved consensus N-terminal region (MKKLKKLKL), a conserved Gly-Gly cleavage motif, after which a Cys-rich-15-residue sequence follows. Interestingly, the identified peptide from BGC1 presented a similar consensus N-terminal sequence (MKKLGKIKL) and a double glycine motif. However, besides the CXCXC motif recognised by rSAM, the identified core peptide sequence was longer than canonical core peptides from the TIGR04149 protein family, consisting of 41 amino acids. Moreover, the predicted core peptide contained a second GG motif in the C-terminal region ( Figure 2B ). The presence of two rSAM-related ORFs in BGC1 suggests the modification of this peptide by these rSAM proteins. In fact, many enzymes of the rSAM superfamily are recruited for PTMs of RiPPs, which usually enhances the peptide’s stability and substrate recognition and/or is critical for their activity.[23–25,29,30,50] BGC1 presented two ORFS possibly involved in peptide modification: a rSAM peptide maturase (HMPREF1007_03210, UniProt: E5VEM4) and a pseudo-rSAM (HMPREF1007_03209, UniProt: E5VEM3). Enzymes from the rSAM superfamily catalyse a wide variety of reactions involving the creation of free radical intermediates. To identify the function of these enzymes in BGC1, both protein sequences were retrieved and used as a query in RadicalSAM.org (https:// radicalsam.org/)[40], a web-based tool developed by Gerlt and co-workers to help in the identification and interpretation of rSAM sequences. The rSAM peptide maturase protein sequence (E5VEM4) diverged from cluster-1-1 at cluster-1-1-4 with an Alignment Score (AS) of 45; however, cluster-1-1-4:45 contains rSAM sequences with low sequence similarity ( Supplementary Figure S1 ). Therefore, a more stringent alignment search was applied (AS=50). This revealed a tight cluster of nodes, suggesting that the encoded proteins are interrelated and could perform the same chemistry ( Supplementary Figure S2 A ). Besides the identified cluster, sequence alignment showed two length peaks ( Supplementary Figure S2B ) belonging to hits with low sequence similarity ( Supplementary Figure S2C ), indicating that E5VEM4 could be further separated. Further analysis of the E5VEM4 protein sequence with increased alignment scores resolved cluster-1-1-556:110 ( Figure 3 ). Genome Neighbourhood analysis revealed that rSAM sequences belonging to cluster-1-1-556:110 were spread between 9 Parabacteroides and 24 Bacteroides species ( Figure 4 ). Moreover, protein alignment revealed a separation from sequences belonging to B. fragilis from B. uniformis , Bacteroides sp. and from Parabacteroides sp. and P. distasonis . All sequences presented the CX 3 CX 2 CX motif ( Supplementary Figure S3A ) and a SPASM/twitch domain albeit interspecific variations ( Supplementary Figure S3B ). While a high AS helps in sorting the query sequence into clusters containing isofunctional rSAMs, there were no annotated protein sequences in the identified cluster-1-1-556:110. Preventing associating a known functionality from previously annotated sequences in the same cluster as E5VEM4. Hence, previous clusters were explored until annotated protein sequences were found. Cluster 1-1-4:45 contained 8 annotated sequences ( Supplementary Table S3 ). Thus, the annotated protein sequences were retrieved and aligned with the two rSAM protein sequences from BGC1 (E5VEM3 and E5VEM4) using MUSCLE ( Figure 5 ). Annotated sequences were labelled as anaerobic sulfatase-maturating enzymes. Previously, other peptide maturase proteins have been described as dual-substrate enzymes involved in the PTM of a cysteine or serine residue in the target sequence.[51,31] Similar rSAM ORFs from the Bacteroidota phylum have been proposed to fulfil a similar activity[52] despite the low sequence similarity between previously annotated rSAM proteins within the cluster-1-14:45 and E5VEM4 ( Supplementary Figure S1 and Figure 5 ). Enzymes containing the SPASM domain have been described to form Cα-S bond formation (AlbA, ThnB, ThrC/C, SkfB), C-C bond formation (Pqqe, StrB), epimerisation (PoyD), and Decarboxylation and C-C bond formation (MftC). We hypothesised that E5VEM4 could perform a similar chemistry as other anaerobic sulfatase-maturating enzymes within the radical SAM cluster, catalysing the PTM modification of a serine or cysteine into a 3-oxoalanine, also known as C(alpha)-formylglycine (FGly). Based on the amino acid sequence of the putative peptide in BGC1, E5VEM4 could belong to the “Ser-type” sulfatase maturation enzyme family. The presence of several serine residues in the absence of cysteine residues outside the recognition motif would align with our hypothesis. However, other maturases such as CteB[50] and Tte1186[53] maturases have been characterised to perform γ-thioether linkages between Cys and Thr residues. The presence of several Cys and Thr residues on the precursor peptide suggests that the rSAM from BGC1 could also form such thioether linkages.[52] Further analysis of the Genome Neighbourhood revealed that out of the 36 clusters identified, 28 clusters showed a 60 to 74 aa ORF that belonged to IPR026408 (GG_sam_targ_CFB) rSAM-modified peptides, supporting the role of rSAM peptide maturases in the modification of RiPPs. Protein alignment of these peptides showed high intraspecific similarity, where peptides from Bacteroides fragilis clustered separately from Parabacteroides species and from Bacteroides sp. and Bacteroides uniformis ( Figure 6 ). Interestingly, while all clusters identified contained at least one rSAM ORF ( Supplementary Figure S3 ), only peptides from the identified Bacteroides sp. and Bacteroides uniformis species showed the CXCXC motif recognised by rSAM. These Gly-rich peptides may form a new subclass of bacteriocins and may be useful in discovering novel strategies for peptide modification and/or microbiota modulation. Leader peptide removal can be coupled with peptide export by some class I lanthipeptides and most class II lanthipeptides,[54] in the form of PCAT enzymes ( Figure 2 ). LanT enzymes are bifunctional proteins containing an N-terminal peptidase C39 domain coupled to a type-1 transmembrane domain and a C-terminal P-loop in charge of nucleoside triphosphate hydrolysis. These ABC transporters cleave the leader peptide from the core peptide at the Gly-Gly motif and transport the peptide across the membrane.[54] However, because of an outer membrane (OM) in Gram-negative bacteria, transport of the cargo from the periplasm to the exterior of the OM requires the presence of additional export proteins. For instance, a sophisticated export mechanism was proposed for pinensins, an anti-fungal peptide proposed by Mohr and colleagues in the Gram-negative Chitinophaga pinensis [55]. The post-translationally modified pinensins are cleaved while exported to the periplasm by a LanT enzyme (PinT). Subsequently, the active peptide is exported through the OM by a TolC-dependent efflux pump[55]. Tripartite efflux pumps have been widely described in the literature,[56–58] and, in fact, a similar cluster architecture is present in BGC1 ( Figure 2 ). In the case of the BGC1 cluster, the LanT encoded by HMPREF1007_03208, downstream the rSAM ORFs, is followed by a periplasmic adaptor HlyD-like encoded by HMPREF1007_03207. Based on the similar cluster architecture, we propose a similar export mechanism: the leader peptide is cleaved, and the core peptide is transported across the membrane by LanT, where the HlyD-like periplasmic adaptor establishes and stabilises the interaction with the OM exporter (TonC). Although we did not identify the TolC OM exporter directly in the BGC1 cluster, the lack of a tonC ORF in BGC1 is consistent with other observations where only the adaptor and transmembrane pump would be present in a cluster[52,56]. TonC can be encoded separately since it has various interactions with other transporters and adaptors, providing functional diversity[57]. Finally, the TonB-dependent receptor (HMPREF1007_03211) could be involved in sensing the extracellular rSAM-modified RiPPs to control and feedback its expression. Furthermore, upstream BGC1, there are five ORFs involved in transport, encoding two drug efflux proteins, one outer membrane efflux protein, an AcrB/AcrD/AcrF family protein and an efflux RND major facilitator protein (MFP), which could alternatively be involved in peptide export through the OM ( Supplementary Figure S4, Figure 7 ). In fact, these transport-related ORFs could also be involved in the transport of two additional putative peptides located upstream. BGC2 contains 21 ORFs, including two sequences belonging to the PF14055 protein family. Protein sequences in this family present a conserved NVEALA motif, NIEALA, as of in peptides from BGC2, preceding a Gly-Gly motif. These peptides also showed a conserved N-terminal region, consistent with the consensus motif from rSAM-modified peptides (TIGR04149 and IPR026408). Moreover, peptide A2 presents the signature motif KXXXW, recognised by a rSAM enzyme from family TIGR04080, which, in Streptococcus thermophilus, catalyses the cyclisation between Lys and Trp.[21,59] However, the putative peptide sequences did not show a CXCXC recognition motif in their core peptide region despite the presence of an rSAM OFR in the cluster. In fact, the rSAM (E5VER0) aligned within the Megacluster-2-4-1: Elongator protein-like ( Supplementary Figure S5 ). These protein clusters showed low sequence similarity, and while there were no annotated protein sequences, one sequence, P0ADW6, was identified as an iron-sulphur protein, [60] which can cleave S-adenosyl-L-methionine into methionine and 5'-deoxyadenosine (AdoH). While the rSAM ORF from BGC2 did not contain the consensus CX 3 CX 2 C or the SPASM/twitch motif, it cannot be ruled out that it could be involved in peptide modification by other mechanisms involving alternative peptide recognition sequences. Interestingly, analysis of the protein sequences from the NVEALA protein family (namely, A1 and A2 from BGC2) by the Domain Architecture Retrieval Tool (CDART) revealed several protein families containing the NVEALA domain followed by either a peptidase M76 domain, a LemA domain, a TolB-like domain or a thioredoxin-like domain, plus an additional protein family containing a rSAM domain followed by a NVEALA domain. This could suggest that these peptides are involved in regulatory functions attached to the N-terminal regions of selected proteins. Biosynthetic cluster analysis of Bacteroides fragilis species reveals a conserved cluster architecture. Genome mining with BAGEL4 of B. fragilis 3_1_12 (Bf1) revealed 2 antimicrobial clusters, BGC3 ( Figure 8A ) and BGC4 ( Figure 8B ). Interestingly, clusters from Bf1 contained peptides with an N-terminal region similar to those present in B6; however, several LanC-like ORFs were present in Bf1. Lanthionine synthetase (LanC) proteins are implicated in the post-translational modification of lantibiotics, and they conform a vast protein family with broad substrate specificity, largely distributed across eukaryotic and prokaryotic phyla.[61] Particularly, in class I lanthipeptides, after the dehydration of the Ser and Thr residues in the core region of the peptide via the lanthionine dehydratase (LanB), the closing of the lanthionine ring is achieved following a Michael-type addition of Cys residues onto the modified Ser and Thr residues.[28,52] Although LanC-like proteins form a vastly diverse group of proteins, they share highly conserved features. In the case of the well-characterised nisin-modifying NisC[62], this enzyme presents a highly conserved triad of residues involved in coordinating the zinc ion: Cys 284 , Cys 330 , and His 331 , as well as two other residues that are conserved among the LanC cyclases: His 212 and Arg 280 . However, LanC sequences from Bf1 showed no conservation in these residues ( Supplementary Figure S6 ). This may suggest that these LanC-like ORFs lack the functionality associated with LanC enzymes in the biosynthetic process of class I lanthipeptides. However, the presence and number of LanC-like ORFs in the different B. fragilis strains in this study ( Supplementary Figure S7 ) and across the Bacteroidota phylum (data not shown) suggest a conserved function. Finally, BGC3 and BGC4 and the various BGCs identified in the other B. fragilis strains used in this study (Supplementary Figure S7) contain one or two rSAM-related ORFs. These rSAM ORFs belong to the same Megacluster-1-1: SPASM/twitch domain containing as the rSAM ORFs from BGC1 ( Figure 5 ). This, together with the presence of the CXCXC rSAM recognition motif in the sequences of the putative antimicrobial peptides from both BGC3 and BGC4, suggests that these rSAM ORFs perform a similar function, catalysing the transformation of a Ser residue to 3-oxoalanine. Biosynthetic cluster analysis fails to reveal antimicrobial clusters in P. melaninogenica D18. Despite the broad antimicrobial capabilities of P. melaninogenica D18, antimicrobial cluster mining of this strain showed no areas of interest in both BAGEL4[37] and PRISM[63]. AntiSMASH[38] analysis also resulted in no regions containing antimicrobial clusters. However, a manual search of regions adjacent to a saccharide cluster resulted in the identification of a potential antimicrobial cluster, BGC5 hereafter ( Figure 9A ). BGC5 encodes 14 open reading frames (ORFs) comprising 1 putative peptide sequence and 3 transporter ORFs. BGC5_A1 showed little sequence similarity to peptide sequences identified in B6 in this work ( Figure 9B ). Hence, to investigate the presence of sequences similar to this putative antimicrobial peptide, its sequence was retrieved as an amino acid FASTA file and similar sequences were mined from genomes deposited in NCBI using BLASTP. A total of 47 amino acid sequences of similar proteins were found within members of the Prevotellaceae family and 1 belonging to Solobacterium sp. from the Bacillota phylum ( Figure 10 ). Peptide sequences presented a similar N-terminal sequence to the consensus MKKLKKLKL sequence and a Gly-Gly motif, followed by a second double Gly motif in some sequences. Despite these similarities, none of these sequences showed the CXCXC rSAM recognition motif. These sequences present a somewhat conserved Gly-Gly motif followed by a conserved PNEKNQDDIDT domain prior to a second conserved Gly-Gly motif ( Figure 10 ). This, together with a highly conserved leader peptide region, suggests that these peptides, present in various Prevotellaceae species, are subjected to PTMs, although their nature remains unknown. Discussion Over the last decade, the amount of scientific evidence about MAFLD and its epidemiology has risen exponentially, highlighting its epidemiology in adults, the recent rise in prevalence in children and adolescents, the impact on life and the economic burden affecting population and societies, particularly during the later stages of the disease.[64–69] While the effect of the bacterial gut microbiota on MAFLD has been established, the oral microbiota has also been proposed to play a role in contributing to the development of metabolic diseases[70] due to its ability to influence the gut microbiota by translocation from the mouth cavity to the gut and/or by metabolite production. Moreover, the antimicrobial potential of the oral microbiota remains understudied. Hence, several strains from the gut and oral origin were tested here to identify antimicrobial activity against members of the two gastrointestinal regions. Our assays revealed intra- and inter-species antimicrobial interactions within the oral microbiota and against selected gut microbiota members. While some strains displayed a wide-spectrum antimicrobial profile, they could not inhibit the growth of known ESKAPE pathogens. However, these results allowed the discovery of novel antimicrobials for targeted microbiome modulation since they could inhibit B. stercoris DSM 19555 and B. salyersiae DSM 18765, strains previously correlated with the progression of MAFLD.[49] Moreover, we and others have described the potential for novel antimicrobial compounds from the Bacteroidota phylum.[52,55,71–78] However, it still remains largely unexplored, despite the widely spread putative RiPP biosynthetic clusters within the Bacteroidota phylum in the human gut microbiota.[52] The putative antimicrobial peptides identified in this study share some similarities despite the differences in modification proteins across the three BGCs: a double Gly-Gly motif, a similar N-terminal region and a cluster architecture flanked by transporter genes, a common feature in other bacteriocins which need to be exported to the outside of the cells. These putative peptides also share a long leader region; however, not as long as in thiazole/oxazole-modified microcins (TOMM) clusters, where up to 83 and 70 for NHLP and N11P residue length leader peptides were found.[79] Finally, these peptides are remarkably conserved across phylogenetically related strains, suggesting they may be necessary in establishing and/or maintaining bacterial presence in the GI tract. Interestingly, several of the identified BGCs contained one or more glycosyltransferase ORFs, suggesting the possibility of their involvement in the PTMs of the peptides. In fact, glycosyltransferases from the IPR026419/TIGR04157 protein family appear to be related to clusters in which rSAM enzymes are present and involved in peptide modification.[59] We proposed a PTM system of the peptide A1 from BGC1 and other peptides from B. uniformis species, where a rSAM catalyses the modification of Ser residues followed by putative glycosylation. The modified peptide is then cleaved and exported by LanT and a TolC-dependent efflux pump. A process similar to the PTMs of the predicted peptides from BGC3 and BGC4. The analysis of peptide sequences from BGC2 showed that these peptides contain a characteristic NIEALA motif, and one of them (BGC2_A2) showed the KXXXW motif, also recognised by other rSAM enzymes (family TIGR04080). These motives are also present in quorum-sensing peptides from Streptococcus thermophilus ,[21] which could shed light on the functionality of the peptides in BGC2. Finally, the prevalence across Prevotella species of similar sequences to the A1 ORF from BGC5 suggests a conserved ecological reason for producing such molecules. However, their production and maturation remain unknown. Unfortunately, other well-described approaches based on neighbouring information could not be applied in this case since we found that genomic information from members of the Bacteroidales order is lacking compared with other taxonomic groups. While the link between the observed antimicrobial activity and the identified BGCs could not be established, no additional BGCs were identified using common mining tools. Which may suggest a causal link between the observed halos and the identified peptides, in particular for the BGC1.The novel gene clusters found provide an entrance to discovering and characterising novel antimicrobial compounds from Bacteroidota. This will require further efforts in their isolation, or heterologous expression, purification and characterisation, which are future plans. The advent of new genome-mining tools[37,38,63,80,81] and the accessibility of genomic databases increased natural product discovery.[78,82–84] However, this did not translate equally to Gram-negative bacteria, which remain still some years behind in terms of mining for natural products. In this work, automated search with genome-mining tools proved inaccurate and incomplete, and only together with manual search could interesting BGCs be identified. This highlights the need for comprehensive knowledge of bacteriocins from Gram-negative bacteria specifically. Furthermore, to gain more insights into the molecular mechanism and role of the peptides identified, ideally, a reliable and species-optimised genetic toolbox for genetic engineering of Bacteroidota should be established. While some progress is underway[85], a Bacteroidota's tailored genetic toolbox will immensely contribute to the fundamental understanding of their ecology in the gut as well as their role in the disease state. Declarations Acknowledgements We thank BEI Resources, NIAID, NIH for providing the bacterial strains listed in Table 1 , obtained as part of the Human Microbiome Project. This work was supported by Marie Sklodowska-Curie Actions (MSCA) and Innovative Training Networks, H2020-MSCA-ITN-2018, grant number: 813781, “BestTreat.” Data availability The authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials. Conflict of interests The authors confirm that no known competing financial interests or personal relationships influenced the work reported in this paper. Author contribution Conceptualization: DGM, MVFC, OPK. Methodology: DGM, MVFC and WM. Software: DGM and WM. Writing-original draft: DGM and WM. Review and editing: DGM, MVFC, OPK. Supervision: OPK. All authors have read and agreed to the published version of the manuscript. References T.N.H.W. Group, J. Peterson, S. Garges, M. Giovanni, P. McInnes, L. 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Supplementary Files SupplementaryFiguresMiningBact.docx Supplementary Figures SupplementaryTablesMiningBact.docx Supplementary Tables Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3875369","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267806179,"identity":"447b778f-eb8e-46c8-9474-adf835fbb2b1","order_by":0,"name":"Diego Garcia-Morena","email":"","orcid":"","institution":"University of Groningen","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Garcia-Morena","suffix":""},{"id":267806180,"identity":"cbd1a537-54f9-4112-a871-5de53e699e87","order_by":1,"name":"Maria Victoria Fernandez-Cantos","email":"","orcid":"","institution":"University of Groningen","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Victoria","lastName":"Fernandez-Cantos","suffix":""},{"id":267806181,"identity":"a8672793-7ded-46d6-96a0-dff50364c3f9","order_by":2,"name":"Willem Maathuis","email":"","orcid":"","institution":"University of Groningen","correspondingAuthor":false,"prefix":"","firstName":"Willem","middleName":"","lastName":"Maathuis","suffix":""},{"id":267806182,"identity":"d8607ab3-02e0-4635-b65a-98a9ed3c8aec","order_by":3,"name":"Oscar Paul Kuipers","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYJACxgYGCTD9AMxlJ0ELswGYy0ycFjBgkyBKCz8D7wHGmW0WefzSx59VV7bVMpgT0iLZwJfAuLFNoliyL8fs5tm24wyWzQS0GBzgMWB82CaRuOEMD9vNxrZjDAaHidWy/wz7s0LitWwE2cLDYMbY2FZDWItkM1/CwRnnJIolzvAYSzacA5pASAs/e+/Bhz1ldXn8PewPPzaU1ckZHG8goIeZh+EAkEqAcg/zEFAPAhA1MC11ROgYBaNgFIyCkQYAEh09LCbJ6QkAAAAASUVORK5CYII=","orcid":"","institution":"University of Groningen","correspondingAuthor":true,"prefix":"","firstName":"Oscar","middleName":"Paul","lastName":"Kuipers","suffix":""}],"badges":[],"createdAt":"2024-01-18 09:38:00","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3875369/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3875369/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50723097,"identity":"d5917845-8b3e-46a5-b282-f9427d730b47","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76359,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the process of antimicrobial assays.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/b3eb6067ef1d1373e6a88247.png"},{"id":50723980,"identity":"b74bd860-2bdd-4e5b-b298-ba54f6aead9b","added_by":"auto","created_at":"2024-02-06 10:37:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Schematic overview of BGC1, including the putative peptide gene in light green, rSAM gene in orange, pseudo rSAM in yellow, hypothetical proteins (hp) in grey and export/resistance genes in blue. \u003cstrong\u003eB)\u003c/strong\u003e The sequence from the putative peptide A1 is shown with colour highlights in the putative core peptide with orange highlights in the Gly residues, Cys in pink, Ser in green and Thr residues in blue.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/b001e7a366c6f9cb2ae39f25.png"},{"id":50723102,"identity":"1031f4d8-e49b-4f4c-9ab8-d67aedc048ae","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eSequence Similarity Network of cluster-1-1-42:50 representing the nodes connecting the different protein sequences present in this cluster. \u003cstrong\u003eB)\u003c/strong\u003e Table with the number of sequence IDs, the convergence ratio (CR) and the conserved Cys residues in the rSAM proteins within cluster-1-1-42:50. C) Protein sequence logo of the sequences within the cluster-1-1-42:50. Conserved Cys residues are highlighted in red.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/4b47efee3cb4ac30103bf1fe.png"},{"id":50723569,"identity":"80cf69d8-4490-48c9-993d-1f5868fc81b7","added_by":"auto","created_at":"2024-02-06 10:29:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89208,"visible":true,"origin":"","legend":"\u003cp\u003eGenome neighbourhood diagram depicting clusters that contain rSAM sequences in the same Sequence Similarity Network as E5VEM4 in cluster 1-1-556:110. Radical SAM proteins belonging to the same cluster as E5VEM4 are positioned in the centre of the diagram, coloured red and in the 5’ to 3’ direction. The colour in each arrow represents a Pfam. Some arrows have multiple colours, representing that the protein is a fusion of multiple families. Clusters with more than 90% identity are grouped together. The number of clusters grouped is indicated under each query.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/bf2723723d0cbc302fe58c35.png"},{"id":50723101,"identity":"c544c465-94ce-4170-aae9-002390a5e3d5","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":216716,"visible":true,"origin":"","legend":"\u003cp\u003eMuscle alignment of rSAM proteins present in BGC1 and annotated rSAM sequences from cluster-1-1-4:45 grouped and sorted by pairwise identity showcasing the CX\u003csup\u003e3\u003c/sup\u003eCX\u003csup\u003e2\u003c/sup\u003eC motif \u003cstrong\u003e(A)\u003c/strong\u003e and the SPASM/twitch domain \u003cstrong\u003e(B)\u003c/strong\u003e C\u003csub\u003e367\u003c/sub\u003eX\u003csup\u003e9–15\u003c/sup\u003eG\u003csub\u003e380\u003c/sub\u003eX\u003csup\u003e4\u003c/sup\u003eC\u003csub\u003e385\u003c/sub\u003e-gap-C\u003csub\u003e424\u003c/sub\u003eX\u003csup\u003e2\u003c/sup\u003eC\u003csub\u003e427\u003c/sub\u003eX\u003csup\u003e5\u003c/sup\u003eC\u003csub\u003e433\u003c/sub\u003eX\u003csup\u003e3\u003c/sup\u003eC\u003csub\u003e436\u003c/sub\u003e-gap-C\u003csub\u003e450\u003c/sub\u003e. Cys residues are highlighted in dark blue, and Gly residues in orange. From top to bottom: A0A378Y5A1_PAEPO: Arylsulfatase regulator from \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e, C3HC15_BACTU: rSAM domain protein from \u003cem\u003eBacillus thuringiensis \u003c/em\u003eserovar huazhongensis,\u0026nbsp; the highlighted sequence names (black) belong to the pseudo-rSAM and rSAM ORFs from \u003cem\u003eBacteroides sp. \u003c/em\u003e4_1_36 (B6), respectively, 1B2Q7_PARDP: rSAM domain protein from \u003cem\u003eParacoccus denitrificans\u003c/em\u003e strain Pd1222, A3DDW1_ACET2: rSAM domain protein from \u003cem\u003eAcetivibrio thermocellus\u003c/em\u003e strain ATCC 27405, Q8RAM6_CALS4: Arylsulfatase regulator (Fe-S oxidoreductase) from \u003cem\u003eCaldanaerobacter subterraneus\u003c/em\u003e subsp. \u003cem\u003etengcongensis\u003c/em\u003e strain DSM 15242, G0LD27_RUMGN: Putative Fe-S oxidoreductase from \u003cem\u003eRuminococcus gnavus\u003c/em\u003e, G0LD12_RUMGN: Putative Fe-S oxidoreductase from \u003cem\u003eRuminococcus gnavus\u003c/em\u003e, and F5AT08_BACTU: Radical SAM domain protein from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/60e63bf3c236bf981e1dfae4.png"},{"id":50723982,"identity":"c6ba2d1e-7ad5-4352-8e9a-90f352ba311c","added_by":"auto","created_at":"2024-02-06 10:37:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":343266,"visible":true,"origin":"","legend":"\u003cp\u003eMuscle alignment of non-redundant peptide sequences identified in the Genome Neighbour Analysis from cluster-1-1-556:110 sorted by pairwise identity. Group sequences belong to\u003cem\u003eBacteroides fragilis \u003c/em\u003e(top), \u003cem\u003eBacteroides uniformis \u003c/em\u003eand \u003cem\u003eBacteroides sp. \u003c/em\u003e(middle), and \u003cem\u003eParabacteroides distasonis \u003c/em\u003eand \u003cem\u003eParabacteroides sp. \u003c/em\u003e(Bottom). Leader regions are coloured by the percentage identity, where darker blue indicates higher conservation across all sequences, while the putative core peptides are depicted showing orange highlights in the Gly residues, Cys in pink, Ser in green and Thr residues in blue. The highlighted sequence name (black) belongs to \u003cem\u003eBacteroides sp. \u003c/em\u003e4_1_36 (B6).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/cd51f0f5c2257e1f779b14a4.png"},{"id":50723573,"identity":"ac346053-2a5b-43b4-9127-9a727f0cf6d5","added_by":"auto","created_at":"2024-02-06 10:29:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71556,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of region GL622507.1 from B6 (BGC2). ORFs depicting the putative peptides are shown in light green, rSAM genes in orange, other biosynthetic genes in burgundy, hypothetical proteins (hp) in grey and export/resistance genes in blue. The sequence from the putative peptides A1 and A2 are shown with colour highlights in the putative core peptides showing orange highlights in the Gly residues, Cys in pink, Ser in green and Thr residues in blue.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/bd1d7b29d8b20ca50a0d4154.png"},{"id":50723571,"identity":"0911c568-a30d-4768-8f9e-3808d1eb38fd","added_by":"auto","created_at":"2024-02-06 10:29:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":82663,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of region EQ9732171.3\u003cstrong\u003e (A)\u003c/strong\u003e and EQ9732181\u003cstrong\u003e (B)\u003c/strong\u003e from Bf1 (BGC3 and BGC4, respectively). ORFs depicting the putative peptides are shown in light green, rSAM genes in orange, other biosynthetic genes in burgundy, hypothetical proteins (hp) in grey and export/resistance genes in blue. The sequence from the putative peptide is shown below their respective BGC with colour highlights in the putative core peptides showing orange highlights in the Gly residues, Cys in pink, Ser in green and Thr residues in blue.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/5e2d929c48281605a7724f2c.png"},{"id":50723107,"identity":"21f8dc17-a37e-4a72-a8d7-670bf3e6f360","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":139714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Schematic overview of BGC3 from \u003cem\u003ePrevotella melaninogenica \u003c/em\u003eD18. ORFs depicting the putative peptides are shown in light green, other biosynthetic genes in burgundy, hypothetical proteins (hp) in grey and export/resistance genes in blue.\u003cstrong\u003e B) \u003c/strong\u003eSequences from the putative peptides from the five BGCs are shown, sorted by pairwise identity and with colour highlights in the core peptides to identify the double Gly motif, the Cys motif present in rSAM peptides, and the Ser and Thr residues commonly modified in lantibiotics. Hence, orange highlights are used for Gly residues, Cys residues are highlighted in pink, Ser in green and Thr in blue. Leader regions are coloured by the percentage identity, where darker blue indicates higher conservation across all sequences.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/36255d7dd9e06068349ff1c2.png"},{"id":50723109,"identity":"fdcbcc34-e7d8-4098-a420-0e97f810bb66","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":516821,"visible":true,"origin":"","legend":"\u003cp\u003eSequences from the putative peptides from BlastP analysis from the putative peptide from \u003cem\u003ePrevotella melaninogenica \u003c/em\u003eD18 are shown with colour highlights in the core peptides showing orange highlights in the Gly residues, Cys in pink, Ser in green and Thr residues in blue. Leader regions are coloured by the percentage identity, where darker blue indicates higher conservation across all sequences. The highlighted sequence names (black) belong to \u003cem\u003ePrevotella melaninogenica \u003c/em\u003eD18 (top) and \u003cem\u003eSolobacterium sp. \u003c/em\u003e(bottom).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/66fa077324f8ec0615f08e77.png"},{"id":50724180,"identity":"927f453c-cf01-4d47-9fcb-924b99375c2f","added_by":"auto","created_at":"2024-02-06 10:45:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2114850,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/9337e887-4a38-4444-b257-7b0d525b5d20.pdf"},{"id":50723574,"identity":"3000a681-2179-4da8-be60-6e5049edddc9","added_by":"auto","created_at":"2024-02-06 10:29:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3438308,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryFiguresMiningBact.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/48dbabf06bfde5e65aa9b446.docx"},{"id":50723099,"identity":"114979c6-1e54-4786-b9ef-767ac0817b46","added_by":"auto","created_at":"2024-02-06 10:21:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28736,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Tables\u003c/p\u003e","description":"","filename":"SupplementaryTablesMiningBact.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/2130ce14ab4204ff9bcd756e.docx"},{"id":50723981,"identity":"46bd3745-77f9-4ca0-a613-031a64a20df4","added_by":"auto","created_at":"2024-02-06 10:37:42","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24459,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875369/v1/75f0cffa82a37802aa9b5ba6.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eAntimicrobial activity screening of Bacteroidota and genome-based analysis of their antimicrobial biosynthetic potential\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlong the gastrointestinal (GI) tract, the presence of (semi)processed nutrients creates an ideal environment for a wide variety of microorganisms to thrive, with some of the most dense bacterial communities found in the human oral cavity and the colon.[1] In both the oral cavity and the intestine, the predominant phyla of bacteria are the Firmicutes and the Bacteroidetes (Bacillota and Bacteroidota, hereafter)[2]. However, the balance between them changes depending on the location in the GI tract.[3\u0026ndash;7]\u003c/p\u003e \u003cp\u003eIt has been shown that bacteria play a vital role in the metabolism and bioavailability of carbohydrates, amino acids, and certain vitamins and the correct development of the immune system.[8] Moreover, the presence of beneficial and neutral bacteria along the GI tract can prevent the establishment of harmful bacteria by outcompeting potential pathogens and excretion of bacterial antimicrobial compounds.[9] However, bacteria in the gut microbiota can also cause various diseases by disturbing the epithelial lining, causing cell death and creating an entrance for bacteria into the bloodstream, affecting proximal and distal organs. Recent research has shown that even without direct signs of cell death, modifications in the microbiota composition are associated with many diseases, such as irritable bowel syndrome (IBS), diabetes, cardiovascular disease and Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as Non-Alcoholic Fatty Liver Disease (NAFLD).[10\u0026ndash;15] However, the bacterial gut microbiota is not the sole driver of disease. The oral microbiota has a major role in oral health, and recent evidence links its impact on systemic health and disease (recently reviewed by Baker et al., 2023).[16]\u003c/p\u003e \u003cp\u003eTo control these large communities of microorganisms, the body maintains a multi-layered defence. The established GI microbiota is also a part of this multi-layered defence, partly controlling its own quantitative and qualitative composition. Bacteria in the GI tract excrete various bacterial antimicrobial compounds to gain a relative advantage over competing species or members of the same genus. Bacteriocins, in particular, are a family of natural products with antimicrobial activity, comprising several families, such as the ribosomally synthesised and post-translationally modified peptides (RiPPs). These peptides present a huge structural variability due to the vast array of post-translational modifications (PTM) generated by enzymes encoded by adjacent genes.[17\u0026ndash;19] Radical-SAM (rSAM) enzymes have been widely reported to be involved in peptide modification, defining post-translational modifications on Epipeptides,[20] Streptide,[21] Mycofactocin,[22,23] Pyrroloquinoline quinone,[24] Ranthipeptides,[25] Ryptides,[26] Sactipeptides[27,28] and Spliceotides.[29] Within the vast landscape of rSAM proteins, an increasing number of enzymes contain the SPASM/twitch domain, initially termed after maturases of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eubtilosin, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003eyrroloquinoline quinone (PQQ), \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003enaerobic \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u003c/span\u003eulfatases, and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eycofactocin.[30] In this family, besides the common CX\u003csub\u003e3\u003c/sub\u003eCX\u003csub\u003e2\u003c/sub\u003eC [4Fe-4S] cluster binding motif in the N-terminal region, members of the SPASM/twitch domain also contain a conserved 7-Cys motif (CX\u003csub\u003e9\u003c/sub\u003e\u0026ndash;\u003csub\u003e15\u003c/sub\u003eGX\u003csub\u003e4\u003c/sub\u003eC-gap-CX\u003csub\u003e2\u003c/sub\u003eCX\u003csub\u003e5\u003c/sub\u003eCX\u003csub\u003e3\u003c/sub\u003eC-gap-C) in the C-terminus.[31,32] This motif is involved in the binding of one (twitch) or two (SPASM) auxiliary [4Fe-4S] clusters.[33\u0026ndash;35] The three conserved cysteine residues in the CX\u003csub\u003e3\u003c/sub\u003eCX\u003csub\u003e2\u003c/sub\u003eC motif bind one iron each from the [4Fe-4S] cluster, leaving the fourth iron as a ligand for \u003cem\u003eS\u003c/em\u003e-adenosylmethionine (SAM), which facilitates the reductive cleavage of SAM rendering methionine and a 5\u0026prime;-deoxyadenosyl radical, which will be used in other enzymatic reactions.[31,35,36]\u003c/p\u003e \u003cp\u003eIntra- and/or inter-specific antimicrobial activity could be used as a gut microbiota modulator to prevent the advent or progression of such aforementioned diseases. The present study explores the antimicrobial potential in strains of the Bacteroidota and Fusobacteriota phyla. To achieve this, antimicrobial assays were performed with gut- and oral-isolated Bacteroidota species. When interactions were found, antimicrobial BGCs were mined and analysed. This revealed potential new peptides in \u003cem\u003eBacteroides sp.\u003c/em\u003e 4_1_36, \u003cem\u003eB. Fragilis\u003c/em\u003e 3_1_12 and in \u003cem\u003ePrevotella melaninogenica\u003c/em\u003e D18 able to aid in targeted microbiome modulation.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eSample collection, bacterial strains and culture conditions\u003c/p\u003e \u003cp\u003eBacterial strains used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, showing the names, shorthand code, source, and sampling origin (if known). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e collects the bacterial pathogenic strains used in the \u0026lsquo;Pathogen interactions\u0026rsquo; experiment. Strains were provided by BEI Resources and the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of all anaerobic strains, showing the full scientific and strain name, the shorthand code used, the source and, if known, the donor's sample location and health status.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScientific name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOrigin, health\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides fragilis\u003c/em\u003e 3_1_12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBf1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides fragilis\u003c/em\u003e CL03T12C07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides fragilis\u003c/em\u003e CL05T00C42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBf3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides fragilis\u003c/em\u003e CL07T00C01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBf4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides fragilis\u003c/em\u003e CL07T12C05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBf5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides salyersiae\u003c/em\u003e CL02T12C01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB. sal2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides sp.\u003c/em\u003e 4_1_36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIleum, Crohn\u0026rsquo;s disease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides stercoris\u003c/em\u003e DSM 19555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB. ster1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides vulgatus\u003c/em\u003e DSM 1447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroides xylanisolvens\u003c/em\u003e DSM 18836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBx2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium gonidiaformans\u003c/em\u003e CMW8396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCMW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBacterial vaginosis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum animalis\u003c/em\u003e D11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, inactive Crohn\u0026rsquo;s disease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum animalis\u003c/em\u003e F0419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e CTI-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTI-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, colonic carcinoma\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e MJR7757B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMJR7757B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVaginal swab, pregnant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum polymorphum\u003c/em\u003e F0401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrachea, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium sp.\u003c/em\u003e CM21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium sp.\u003c/em\u003e CM22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCM22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium sp.\u003c/em\u003e F0437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium sp.\u003c/em\u003e OBRC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOBRC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium ulcerans\u003c/em\u003e 12_1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12_1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, Crohn\u0026rsquo;s disease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParabacteroides distasonis\u003c/em\u003e 31_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePar1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParabacteroides goldsteinii\u003c/em\u003e CC87F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePargo1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParabacteroides johnsonii\u003c/em\u003e CL02T12C29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParjo1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParabacteroides merdae\u003c/em\u003e CL03T12C32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParme1/P1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParabacteroides sp.\u003c/em\u003e D13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, ulcerative colitis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhocaeicola dorei\u003c/em\u003e 5_1_36/D4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIleum, Crohn\u0026rsquo;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhocaeicola dorei\u003c/em\u003e CL02T00C15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhocaeicola dorei\u003c/em\u003e CL02T12C06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhocaeicola dorei\u003c/em\u003e CL03T12C01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFaeces, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e F0185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, periodontitis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePorphyromonas sp.\u003c/em\u003e KLE1280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKLE1280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, plaque\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePorphyromonas sp.\u003c/em\u003e W7784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eW7784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, plaque\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella buccae\u003c/em\u003e D17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella denticola\u003c/em\u003e F0289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella histicola\u003c/em\u003e F0411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF0411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, plaque\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella melaninogenica\u003c/em\u003e D18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity, healthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella nigrescens\u003c/em\u003e CC14M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC14M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella oralis\u003c/em\u003e CC98A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCC98A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColon, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella oralis\u003c/em\u003e HGA0225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColonic mucosa, unknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e AL-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e ASLM/F0460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e H1-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e H22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e OTK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTreponema denticola\u003c/em\u003e US-trep/F0459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOral cavity,\u003c/p\u003e \u003cp\u003eperiodontal pocket\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll anaerobic strains were grown at anaerobic conditions in a Coy Anaerobic chamber. The atmospheric conditions were set to 1.5-2.0% H\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e and 90+% N\u003csub\u003e2\u003c/sub\u003e. Strains were grown at 37\u0026deg;C without shaking.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eParabacteroides\u003c/em\u003e, and \u003cem\u003ePhocaeicola\u003c/em\u003e strains were eventually grown in Gifu Anaerobic Broth from HiMedia (GAM; EWC Diagnostics Trade BV) supplemented with 5 \u0026micro;g/mL hemin and 2.5 \u0026micro;g/mL vitamin K1. The supplemented GAM was named GAM complete, or GAM\u003csub\u003ec\u003c/sub\u003e hereafter. \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003ePorphyromonas\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e strains were grown in Tryptone Soya Broth (TSB; Boom B.V.) supplemented with 250 \u0026micro;g/mL hemin and 2.5 \u0026micro;g/mL vitamin K1. The supplemented TSB was named TSB hemin 50x, or TSB\u003csub\u003eh50\u003c/sub\u003e hereafter. GAM\u003csub\u003ec\u003c/sub\u003e and TSB\u003csub\u003eh50\u003c/sub\u003e agar plates were prepared by adding 1.5% agar to the respective broth before autoclaving. Soft agar plates were prepared by adding 0.7% agar to each respective broth before autoclaving. In all cases, supplementation was done after autoclaving. All liquid media were introduced in the anaerobic chamber overnight; agar plates were cast right after supplementation and placed inside the COY anaerobic chamber overnight. Soft agar, autoclaved just before use, was allowed to degasify for 2 min under 20mm Hg of vacuum before being introduced into the anaerobic chamber and then supplemented inside.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of all aerobic strains used, showing the full scientific and strain name and the shorthand code used.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScientific name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e WT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePsa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e LMG 20218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalmonella enterica\u003c/em\u003e LMG 07233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSe\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e LMG 01041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e LMG 15862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e WT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEnterobacter aerogenes\u003c/em\u003e LMG 02094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEnterobacter cloacae\u003c/em\u003e LMG 02783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eScreening of antimicrobial activity\u003c/p\u003e \u003cp\u003eColony antimicrobial assays:\u003c/p\u003e \u003cp\u003eTo find antimicrobial interactions between Bacteroidota species, GAM\u003csub\u003ec\u003c/sub\u003e and TSB\u003csub\u003eh50\u003c/sub\u003e agar plates were poured and spotted with 10\u0026ndash;15 different strains each. The following day, soft (0.7%) agar of the corresponding base media was prepared. Each spotted plate was assigned one of the spotted strains for its overlay, consisting of 1 mL liquid culture mixed into 14 mL 0.7% agar. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, on the third day, the plates were checked for halos, indicating antimicrobial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePathogen interactions:\u003c/p\u003e \u003cp\u003eTo study the potential role of Bacteroidota strains in preventing infections caused by common pathogenic bacterial strains, an interaction assay was performed on a 96-well plate. 8 pathogenic strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were chosen and grown overnight in 4 mL of LB media at 37\u0026deg;C and 220 rpm. The pathogenic strains were diluted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0005 in fresh LB, and 100 \u0026micro;L was added to the corresponding wells in the 96-well plate. 5 Bacteroidota strains were chosen for this experiment, namely B6, Bd2, Bf6, D18, and Parme1. The supernatant of these strains was obtained twice by centrifugation for 3 min at 12,000 rpm. 10 \u0026micro;L of an overnight culture of the pathogenic strains was used to inoculate respective soft agar tubes. On top of an agar layer, 5 mm metal rings were placed, and the inoculated overlay was poured around these rings. After removing the rings, the wells formed were filled with 75 \u0026micro;L of the centrifuged supernatant. For the positive and negative control, 50 \u0026micro;L of fresh GAMc was added in place of Bacteroidota supernatant. The 96-well plate was incubated overnight at 37\u0026deg;C and visually checked the next day.\u003c/p\u003e \u003cp\u003eAntimicrobial compounds mining and metabolite biosynthetic gene cluster analysis\u003c/p\u003e \u003cp\u003eTo identify potential candidate RiPP BGCs, genome assemblies of the candidate strains were obtained from NCBI and the bioinformatic tools BAGEL4[37] and AntiSMASH[38] were used. The identification process involved considering the presence and arrangement of PTM enzymes, particularly rSAM ORFs, putative precursor peptides, and transporters within the area of interest (AOI) from BAGEL4 or the identified clusters from AntiSMASH. The selected AOIs, usually comprising over 20kbs sequence including the detected features, were manually investigated using the InterPro web-based tool,[39] Domain Architecture Retrieval Tool (CDART) and protein\u0026ndash;protein BLAST in the NCBI web service. For rSAM protein clustering and identification, as well as for Genome Neighbouring Analysis, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://radicalsam.org/\u003c/span\u003e\u003cspan address=\"https://radicalsam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[40] was used. Finally, the amino acid sequence of relevant enzymes was obtained using their UniProt IDs and aligned in Jalview 2.11.2.7[41] using MUSCLE[42] on default parameters. Jalview 2.11.2.7 was also used for multiple sequence alignment (MSA) visualisation and Logo generation.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eModified TSB supports the growth of several bacterial strains from the oral microbiota.\u003c/h2\u003e\n\u003cp\u003eWhile GAM\u003csub\u003ec\u0026nbsp;\u003c/sub\u003ewas a suitable media for \u003cem\u003eBacteroides\u003c/em\u003e strains, overnight inocula of \u003cem\u003eFusobacterium, Porphyromonas, Prevotella\u003c/em\u003e and \u003cem\u003eTreponema\u0026nbsp;\u003c/em\u003estrains in GAM\u003csub\u003ec\u003c/sub\u003e resulted in undetectable growth (\u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e). To improve the growth conditions, TSB medium was selected, supplemented with 5 \u0026micro;g/mL hemin and 2.5 \u0026micro;g/mL vitamin K1. This enhanced the growth of several \u003cem\u003eFusobacterium\u003c/em\u003e species. However, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003ePorphyromonas, and Treponema species could still not grow on this medium\u003c/em\u003e. Therefore, TSB supplemented with increased concentrations of hemin was tested, as hemin is an essential growth factor for many Bacteroidota species.[43,44]\u0026nbsp;This proved successful for \u003cem\u003ePrevotella\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePorphyromonas\u0026nbsp;\u003c/em\u003especies\u003cem\u003e,\u0026nbsp;\u003c/em\u003ewhich grew reliably in TSB supplemented with 500 \u0026micro;g/mL hemin and 2.5 \u0026micro;g/mL vitamin K1, but not for \u003cem\u003eTreponema\u0026nbsp;\u003c/em\u003especies (\u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e). In fact, \u003cem\u003eTreponema\u003c/em\u003e was not able to grow in TSB\u003csub\u003eh50\u003c/sub\u003e supplemented with L-cysteine, which can function as an oxygen scavenger, pulling oxygen out of the solution.[45,46]\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAntimicrobial interactions\u003c/h2\u003e\n\u003cp\u003eAntimicrobial tests were conducted in TSB\u003csub\u003eh50\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eTable 3)\u0026nbsp;\u003c/strong\u003eand GAMc (Table 4) using bacterial microbiota strains as producers and sensitive strains. The colony antimicrobial assays revealed that in the conditions studied, \u003cem\u003eBacteroides sp.\u0026nbsp;\u003c/em\u003e4_1_36 (B6) showed antimicrobial activity against members of the \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003ePorphyromonas\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroides\u0026nbsp;\u003c/em\u003egenera but not against the tested \u003cem\u003ePrevotella\u0026nbsp;\u003c/em\u003especies (\u003cstrong\u003eTables 3 \u0026amp; 4\u003c/strong\u003e). On the other hand, under these conditions, \u003cem\u003ePrevotella melaninogenica\u003c/em\u003e D18, originally isolated from an oral swab from a healthy male patient in Canada[47] inhibited the growth of 10 other strains, including members of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003ePorphyromonas\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroides\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eTable 3\u003c/strong\u003e). In particular, D18 showed antimicrobial activity against five other oral strains and four gut strains. Interestingly, two of those five gut strains were: \u003cem\u003eB. stercoris\u0026nbsp;\u003c/em\u003eDSM 19555 (Bster1) and \u003cem\u003eB. salyersiae\u0026nbsp;\u003c/em\u003eCL02T12C01 (Bsal1). Finally, \u003cem\u003eB. fragilis\u0026nbsp;\u003c/em\u003e3_1_12 (Bf1) showed intra-species antimicrobial activity,[48] inhibiting the growth of other \u003cem\u003eB. fragilis\u0026nbsp;\u003c/em\u003estrains, while unable to inhibit the growth of other members of the same genus, except for \u003cem\u003eB. stercoris\u0026nbsp;\u003c/em\u003eDSM 19555 (\u003cstrong\u003eTable 4\u003c/strong\u003e). The antimicrobial activity found in B6, D18 and Bf1 against \u003cem\u003eB. stercoris\u0026nbsp;\u003c/em\u003eDSM 19555 and \u003cem\u003eB. salyersiae\u0026nbsp;\u003c/em\u003eCL02T12C01 is very relevant since these species had been correlated to the progress of MAFLD.[49] Therefore, finding bacterial strains able to impair their growth might prove helpful in developing new ways of dealing with MAFLD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the results from the antimicrobial assays, several strains were selected to further study their influence against known pathogens. However, the antimicrobial test showed no inhibitory effect (\u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe antimicrobial screenings (\u003cstrong\u003eTables 3 \u0026amp; 4\u003c/strong\u003e) revealed \u003cem\u003eP. melaninogenica\u003c/em\u003e D18 as a strain with broad-spectrum antimicrobial activity, and both \u003cem\u003eB. fragilis\u0026nbsp;\u003c/em\u003e3_1_12 (Bf1) and \u003cem\u003eBacteroides sp.\u0026nbsp;\u003c/em\u003e4_1_36 (B6) as strains with a narrower antimicrobial spectrum. Moreover, all three strains showed activity against \u003cem\u003eB. stercoris\u003c/em\u003e DSM 19555, a strain related to MAFLD progression.[49]\u0026nbsp;Therefore, these strains were selected for antimicrobial cluster mining to identify the putative biosynthetic gene cluster (BGC) involved in the halo formation.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eBiosynthetic gene cluster analysis reveals two novel putative antimicrobial clusters in \u003cem\u003eBacteroides sp\u003c/em\u003e. 4_1_36\u003c/h2\u003e\n\u003cp\u003eGenome mining of B6 with BAGEL4 revealed one BGC of interest (\u003cstrong\u003eFigure 2A\u003c/strong\u003e), termed BGC1 hereafter. \u0026nbsp;BGC1 encodes 17 ORFs, including one sequence predicted to be rSAM-modified_RiPP_057 (A1), which belongs to the TIGR04149 protein family or peptides associated with peptide-modifying radical SAM enzymes. Peptides in this family present a characteristic modular sequence, including a leader sequence with a conserved consensus N-terminal region (MKKLKKLKL), a conserved Gly-Gly cleavage motif, after which a Cys-rich-15-residue sequence follows. Interestingly, the identified peptide from BGC1 presented a similar consensus N-terminal sequence (MKKLGKIKL) and a double glycine motif. However, besides the CXCXC motif recognised by rSAM, the identified core peptide sequence was longer than canonical core peptides from the TIGR04149 protein family, consisting of 41 amino acids. Moreover, the predicted core peptide contained a second GG motif in the C-terminal region (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). The presence of two rSAM-related ORFs in BGC1 suggests the modification of this peptide by these rSAM proteins. In fact, many enzymes of the rSAM superfamily are recruited for PTMs of RiPPs, which usually enhances the peptide\u0026rsquo;s stability and substrate recognition and/or is critical for their activity.[23\u0026ndash;25,29,30,50]\u003c/p\u003e\n\u003cp\u003eBGC1 presented two ORFS possibly involved in peptide modification: a rSAM peptide maturase (HMPREF1007_03210, UniProt: E5VEM4)\u0026nbsp;and a pseudo-rSAM (HMPREF1007_03209, UniProt: E5VEM3). Enzymes from the rSAM superfamily catalyse a wide variety of reactions involving the creation of free radical intermediates. To identify the function of these enzymes in BGC1, both protein sequences were retrieved and used as a query in RadicalSAM.org (https:// radicalsam.org/)[40], a web-based tool developed by Gerlt and co-workers to help in the identification and interpretation of rSAM sequences.\u0026nbsp;The rSAM peptide maturase protein sequence (E5VEM4) diverged from\u0026nbsp;cluster-1-1 at cluster-1-1-4 with an Alignment Score (AS) of 45; however, cluster-1-1-4:45 contains rSAM sequences with low sequence similarity (\u003cstrong\u003eSupplementary Figure S1\u003c/strong\u003e).\u0026nbsp;Therefore, a more stringent alignment search was applied (AS=50). This revealed a tight cluster of nodes, suggesting that the encoded proteins are interrelated and could perform the same chemistry (\u003cstrong\u003eSupplementary Figure S2\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e). Besides the identified cluster, sequence alignment showed two length peaks\u0026nbsp;(\u003cstrong\u003eSupplementary Figure S2B\u003c/strong\u003e)\u0026nbsp;belonging to hits with low sequence similarity\u0026nbsp;(\u003cstrong\u003eSupplementary Figure S2C\u003c/strong\u003e), indicating that E5VEM4 could be further separated.\u003c/p\u003e\n\u003cp\u003eFurther analysis of the E5VEM4 protein sequence with increased alignment scores resolved cluster-1-1-556:110 (\u003cstrong\u003eFigure 3\u003c/strong\u003e). Genome Neighbourhood analysis revealed that rSAM sequences belonging to cluster-1-1-556:110 were spread between 9 \u003cem\u003eParabacteroides\u003c/em\u003e and 24 \u003cem\u003eBacteroides\u0026nbsp;\u003c/em\u003especies (\u003cstrong\u003eFigure 4\u003c/strong\u003e). Moreover, protein alignment revealed a separation from sequences belonging to \u003cem\u003eB. fragilis\u003c/em\u003e from \u003cem\u003eB. uniformis\u003c/em\u003e, \u003cem\u003eBacteroides sp.\u0026nbsp;\u003c/em\u003eand from \u003cem\u003eParabacteroides sp.\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. distasonis\u003c/em\u003e. All sequences presented the CX\u003csub\u003e3\u003c/sub\u003eCX\u003csub\u003e2\u003c/sub\u003eCX motif (\u003cstrong\u003eSupplementary Figure S3A\u003c/strong\u003e) and a SPASM/twitch domain albeit interspecific variations (\u003cstrong\u003eSupplementary Figure S3B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWhile\u0026nbsp;a high AS helps in sorting the query sequence into clusters containing isofunctional rSAMs, there were no annotated protein sequences in the identified cluster-1-1-556:110. Preventing associating a known functionality from previously annotated sequences in the same cluster as E5VEM4. Hence, previous clusters were explored until annotated protein sequences were found. Cluster 1-1-4:45 contained 8 annotated sequences (\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e). Thus, the annotated protein sequences were retrieved and aligned with the two rSAM protein sequences from BGC1 (E5VEM3 and E5VEM4) using MUSCLE (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnnotated sequences were labelled as anaerobic sulfatase-maturating enzymes. Previously, other peptide maturase proteins have been described as dual-substrate enzymes involved in the PTM of a cysteine or serine residue in the target sequence.[51,31]\u0026nbsp;Similar rSAM ORFs from the Bacteroidota phylum have been proposed to fulfil a similar activity[52]\u0026nbsp;despite the low\u0026nbsp;sequence similarity between previously annotated rSAM proteins within the cluster-1-14:45 and E5VEM4 (\u003cstrong\u003eSupplementary Figure S1\u003c/strong\u003e and \u003cstrong\u003eFigure 5\u003c/strong\u003e). Enzymes containing the SPASM domain have been described to form C\u0026alpha;-S bond formation (AlbA, ThnB, ThrC/C, SkfB), C-C bond formation (Pqqe, StrB), epimerisation (PoyD), and Decarboxylation and C-C bond formation (MftC). We hypothesised that E5VEM4 could perform a similar chemistry as other anaerobic sulfatase-maturating enzymes within the radical SAM cluster, catalysing the PTM modification of a serine or cysteine into a\u0026nbsp;3-oxoalanine, also known as C(alpha)-formylglycine (FGly). Based on the amino acid sequence of the putative peptide in BGC1, E5VEM4 could belong to the \u0026ldquo;Ser-type\u0026rdquo; sulfatase maturation enzyme family. The presence of several serine residues in the absence of cysteine residues outside the recognition motif would align with our hypothesis. However, other maturases such as CteB[50]\u0026nbsp;and Tte1186[53]\u0026nbsp;maturases have been characterised to perform \u0026gamma;-thioether linkages between Cys and Thr residues. The presence of several Cys and Thr residues on the precursor peptide suggests that the rSAM from BGC1 could also form such thioether linkages.[52]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther analysis of the Genome Neighbourhood revealed that out of the 36 clusters identified, 28 clusters showed a 60 to 74 aa ORF that belonged to IPR026408 (GG_sam_targ_CFB) rSAM-modified peptides, supporting the role of rSAM peptide maturases in the modification of RiPPs. Protein alignment of these peptides showed high intraspecific similarity, where peptides from \u003cem\u003eBacteroides fragilis\u003c/em\u003e clustered separately from \u003cem\u003eParabacteroides\u0026nbsp;\u003c/em\u003especies and from \u003cem\u003eBacteroides sp.\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroides uniformis\u003c/em\u003e (\u003cstrong\u003eFigure 6\u003c/strong\u003e). Interestingly, while all clusters identified contained at least one rSAM ORF (\u003cstrong\u003eSupplementary Figure S3\u003c/strong\u003e), only peptides from the identified \u003cem\u003eBacteroides sp.\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroides uniformis\u0026nbsp;\u003c/em\u003especies showed the CXCXC motif recognised by rSAM. These Gly-rich peptides may form a new subclass of bacteriocins and may be useful in discovering novel strategies for peptide modification and/or microbiota modulation.\u003c/p\u003e\n\u003cp\u003eLeader peptide removal can be coupled with peptide export by some class I lanthipeptides and most class II lanthipeptides,[54]\u0026nbsp;in the form of PCAT enzymes (\u003cstrong\u003eFigure 2\u003c/strong\u003e). \u0026nbsp;LanT enzymes are bifunctional proteins containing an N-terminal peptidase C39 domain coupled to a type-1 transmembrane domain and a C-terminal P-loop in charge of nucleoside triphosphate hydrolysis. These ABC transporters cleave the leader peptide from the core peptide at the Gly-Gly motif and transport the peptide across the membrane.[54] However, because of an outer membrane (OM) in Gram-negative bacteria, transport of the cargo from the periplasm to the exterior of the OM requires the presence of additional export proteins. For instance, a sophisticated export mechanism was proposed for pinensins, an anti-fungal peptide proposed by Mohr and colleagues in the Gram-negative \u003cem\u003eChitinophaga pinensis\u003c/em\u003e[55]. The post-translationally modified pinensins are cleaved while exported to the periplasm by a LanT enzyme (PinT). Subsequently, the active peptide is exported through the OM by a TolC-dependent efflux pump[55]. Tripartite efflux pumps have been widely described in the literature,[56\u0026ndash;58]\u0026nbsp;and, in fact, a similar cluster architecture is present in BGC1 (\u003cstrong\u003eFigure 2\u003c/strong\u003e). In the case of the BGC1 cluster, the LanT encoded by HMPREF1007_03208, downstream the rSAM ORFs, is followed by a periplasmic adaptor HlyD-like encoded by HMPREF1007_03207. Based on the similar cluster architecture, we propose a similar export mechanism: the leader peptide is cleaved, and the core peptide is transported across the membrane by LanT, where the HlyD-like periplasmic adaptor establishes and stabilises the interaction with the OM exporter (TonC). Although we did not identify the TolC OM exporter directly in the BGC1 cluster, the lack of a \u003cem\u003etonC\u0026nbsp;\u003c/em\u003eORF in BGC1 is consistent with other observations where only the adaptor and transmembrane pump would be present in a cluster[52,56]. TonC can be encoded separately since it has various interactions with other transporters and adaptors, providing functional diversity[57]. Finally, the TonB-dependent receptor (HMPREF1007_03211) could be involved in sensing the extracellular rSAM-modified RiPPs to control and feedback its expression. Furthermore, upstream BGC1, there are five ORFs involved in transport, encoding two drug efflux proteins, one outer membrane efflux protein, an AcrB/AcrD/AcrF family protein and an efflux RND major facilitator protein (MFP), which could alternatively be involved in peptide export through the OM (\u003cstrong\u003eSupplementary Figure S4, Figure 7\u003c/strong\u003e). In fact, these transport-related ORFs could also be involved in the transport of two additional putative peptides located upstream.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBGC2 contains 21 ORFs, including two sequences belonging to the\u0026nbsp;PF14055\u0026nbsp;protein family. Protein sequences in this family present a conserved\u0026nbsp;NVEALA\u0026nbsp;motif, NIEALA, as of in peptides from BGC2, preceding a Gly-Gly motif. These peptides also showed a conserved N-terminal region, consistent with the consensus motif from rSAM-modified peptides (TIGR04149 and IPR026408). Moreover, peptide A2 presents the signature motif KXXXW, recognised by a rSAM enzyme from family TIGR04080, which, in\u003cem\u003e\u0026nbsp;Streptococcus thermophilus,\u003c/em\u003e catalyses the cyclisation between Lys and Trp.[21,59]\u0026nbsp;However, the putative peptide sequences did not show a CXCXC recognition motif in their core peptide region despite the presence of an rSAM OFR in the cluster. In fact, the rSAM\u0026nbsp;(E5VER0) aligned within the Megacluster-2-4-1: Elongator protein-like (\u003cstrong\u003eSupplementary Figure S5\u003c/strong\u003e). These protein clusters showed low sequence similarity, and while there were no annotated protein sequences, one sequence,\u0026nbsp;P0ADW6,\u0026nbsp;was identified as an iron-sulphur protein,\u0026nbsp;[60]\u0026nbsp;which\u0026nbsp;can cleave S-adenosyl-L-methionine into methionine and 5\u0026apos;-deoxyadenosine (AdoH). While the rSAM ORF from BGC2 did not contain the consensus CX\u003csub\u003e3\u003c/sub\u003eCX\u003csub\u003e2\u003c/sub\u003eC or the SPASM/twitch motif, it cannot be ruled out that it could be involved in peptide modification by other mechanisms involving alternative peptide recognition sequences.\u003c/p\u003e\n\u003cp\u003eInterestingly, analysis of the protein sequences from the NVEALA protein family (namely, A1 and A2 from BGC2) by the Domain Architecture Retrieval Tool (CDART) revealed several protein families containing the NVEALA domain followed by either a peptidase M76 domain, a LemA domain, a TolB-like domain or a thioredoxin-like domain, plus an additional protein family containing a rSAM domain followed by a NVEALA domain. This could suggest that these peptides are involved in regulatory functions attached to the N-terminal regions of selected proteins.\u003c/p\u003e\n\u003ch2\u003eBiosynthetic cluster analysis of \u003cem\u003eBacteroides fragilis\u0026nbsp;\u003c/em\u003especies reveals a conserved cluster architecture.\u003c/h2\u003e\n\u003cp\u003eGenome mining with BAGEL4 of \u003cem\u003eB. fragilis\u0026nbsp;\u003c/em\u003e3_1_12 (Bf1) revealed 2 antimicrobial clusters, BGC3 (\u003cstrong\u003eFigure 8A\u003c/strong\u003e) and BGC4 (\u003cstrong\u003eFigure 8B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eInterestingly, clusters from Bf1 contained peptides with an N-terminal region similar to those present in B6; however, several LanC-like ORFs were present in Bf1.\u0026nbsp;Lanthionine synthetase (LanC) proteins are implicated in the post-translational modification of lantibiotics, and\u0026nbsp;they conform a vast protein family with broad substrate specificity, largely distributed across eukaryotic and prokaryotic phyla.[61]\u0026nbsp;Particularly, in class I lanthipeptides, after the dehydration of the Ser and Thr residues in the core region of the peptide via the lanthionine dehydratase (LanB), the closing of the lanthionine ring is achieved following a Michael-type addition of Cys residues onto the modified Ser and Thr residues.[28,52]\u0026nbsp;Although LanC-like proteins form a vastly diverse group of proteins, they share highly conserved features. In the case of the well-characterised nisin-modifying NisC[62], this enzyme presents a highly conserved triad of residues involved in coordinating the zinc ion: Cys\u003csub\u003e284\u003c/sub\u003e, Cys\u003csub\u003e330\u003c/sub\u003e, and His\u003csub\u003e331\u003c/sub\u003e, as well as two other residues that are conserved among the LanC cyclases: His\u003csub\u003e212\u003c/sub\u003e and Arg\u003csub\u003e280\u003c/sub\u003e. However, LanC sequences from Bf1 showed no conservation in these residues (\u003cstrong\u003eSupplementary Figure S6\u003c/strong\u003e). This may suggest that these LanC-like ORFs lack the functionality associated with LanC enzymes in the biosynthetic process of class I lanthipeptides. However, the presence and number of LanC-like ORFs in the different \u003cem\u003eB. fragilis\u0026nbsp;\u003c/em\u003estrains in this study (\u003cstrong\u003eSupplementary Figure S7\u003c/strong\u003e) and across the Bacteroidota phylum (data not shown) suggest a conserved function.\u003c/p\u003e\n\u003cp\u003eFinally, BGC3 and BGC4 and the various BGCs identified in the other B. fragilis strains used in this study (Supplementary Figure S7) contain one or two rSAM-related ORFs. These rSAM ORFs belong to the same Megacluster-1-1: SPASM/twitch domain containing as the rSAM ORFs from BGC1 (\u003cstrong\u003eFigure 5\u003c/strong\u003e). This, together with the presence of the CXCXC rSAM recognition motif in the sequences of the putative antimicrobial peptides from both BGC3 and BGC4, suggests that these rSAM ORFs perform a similar function, catalysing the transformation of a Ser residue to 3-oxoalanine.\u003c/p\u003e\n\u003ch2\u003eBiosynthetic cluster analysis fails to reveal antimicrobial clusters in \u003cem\u003eP. melaninogenica\u0026nbsp;\u003c/em\u003eD18.\u003c/h2\u003e\n\u003cp\u003eDespite the broad antimicrobial capabilities of \u003cem\u003eP. melaninogenica\u0026nbsp;\u003c/em\u003eD18, antimicrobial cluster mining of this strain showed no areas of interest in both BAGEL4[37]\u0026nbsp;and PRISM[63]. AntiSMASH[38]\u0026nbsp;analysis also resulted in no regions containing antimicrobial clusters. However, a manual search of regions adjacent to a saccharide cluster resulted in the identification of a potential antimicrobial cluster, BGC5 hereafter (\u003cstrong\u003eFigure 9A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBGC5 encodes 14 open reading frames (ORFs) comprising 1 putative peptide sequence and 3 transporter ORFs. BGC5_A1 showed little sequence similarity to peptide sequences identified in B6 in this work (\u003cstrong\u003eFigure 9B\u003c/strong\u003e). Hence, to investigate the presence of sequences similar to this putative antimicrobial peptide, its sequence was retrieved as an amino acid FASTA file and similar sequences were mined from genomes deposited in NCBI using BLASTP.\u0026nbsp;A total of 47 amino acid sequences of similar proteins were found within members of the Prevotellaceae family and 1 belonging to \u003cem\u003eSolobacterium sp.\u003c/em\u003e from the Bacillota phylum (\u003cstrong\u003eFigure 10\u003c/strong\u003e). Peptide sequences presented a similar N-terminal sequence to the consensus\u0026nbsp;MKKLKKLKL sequence and a Gly-Gly motif, followed by a second double Gly motif in some sequences. Despite these similarities, none of these sequences showed the CXCXC rSAM recognition motif. These sequences present a somewhat conserved Gly-Gly motif followed by a conserved PNEKNQDDIDT domain prior to a second conserved Gly-Gly motif (\u003cstrong\u003eFigure 10\u003c/strong\u003e). This, together with a highly conserved leader peptide region, suggests that these peptides, present in various Prevotellaceae species, are subjected to PTMs, although their nature remains unknown.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the last decade, the amount of scientific evidence about MAFLD and its epidemiology has risen exponentially, highlighting its epidemiology in adults, the recent rise in prevalence in children and adolescents, the impact on life and the economic burden affecting population and societies, particularly during the later stages of the disease.[64\u0026ndash;69] While the effect of the bacterial gut microbiota on MAFLD has been established, the oral microbiota has also been proposed to play a role in contributing to the development of metabolic diseases[70] due to its ability to influence the gut microbiota by translocation from the mouth cavity to the gut and/or by metabolite production. Moreover, the antimicrobial potential of the oral microbiota remains understudied. Hence, several strains from the gut and oral origin were tested here to identify antimicrobial activity against members of the two gastrointestinal regions.\u003c/p\u003e \u003cp\u003eOur assays revealed intra- and inter-species antimicrobial interactions within the oral microbiota and against selected gut microbiota members. While some strains displayed a wide-spectrum antimicrobial profile, they could not inhibit the growth of known ESKAPE pathogens. However, these results allowed the discovery of novel antimicrobials for targeted microbiome modulation since they could inhibit \u003cem\u003eB. stercoris\u003c/em\u003e DSM 19555 and \u003cem\u003eB. salyersiae\u003c/em\u003e DSM 18765, strains previously correlated with the progression of MAFLD.[49] Moreover, we and others have described the potential for novel antimicrobial compounds from the Bacteroidota phylum.[52,55,71\u0026ndash;78] However, it still remains largely unexplored, despite the widely spread putative RiPP biosynthetic clusters within the Bacteroidota phylum in the human gut microbiota.[52]\u003c/p\u003e \u003cp\u003eThe putative antimicrobial peptides identified in this study share some similarities despite the differences in modification proteins across the three BGCs: a double Gly-Gly motif, a similar N-terminal region and a cluster architecture flanked by transporter genes, a common feature in other bacteriocins which need to be exported to the outside of the cells. These putative peptides also share a long leader region; however, not as long as in thiazole/oxazole-modified microcins (TOMM) clusters, where up to 83 and 70 for NHLP and N11P residue length leader peptides were found.[79] Finally, these peptides are remarkably conserved across phylogenetically related strains, suggesting they may be necessary in establishing and/or maintaining bacterial presence in the GI tract. Interestingly, several of the identified BGCs contained one or more glycosyltransferase ORFs, suggesting the possibility of their involvement in the PTMs of the peptides. In fact, glycosyltransferases from the IPR026419/TIGR04157 protein family appear to be related to clusters in which rSAM enzymes are present and involved in peptide modification.[59] We proposed a PTM system of the peptide A1 from BGC1 and other peptides from \u003cem\u003eB. uniformis\u003c/em\u003e species, where a rSAM catalyses the modification of Ser residues followed by putative glycosylation. The modified peptide is then cleaved and exported by LanT and a TolC-dependent efflux pump. A process similar to the PTMs of the predicted peptides from BGC3 and BGC4.\u003c/p\u003e \u003cp\u003eThe analysis of peptide sequences from BGC2 showed that these peptides contain a characteristic NIEALA motif, and one of them (BGC2_A2) showed the KXXXW motif, also recognised by other rSAM enzymes (family TIGR04080). These motives are also present in quorum-sensing peptides from \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e,[21] which could shed light on the functionality of the peptides in BGC2. Finally, the prevalence across \u003cem\u003ePrevotella\u003c/em\u003e species of similar sequences to the A1 ORF from BGC5 suggests a conserved ecological reason for producing such molecules. However, their production and maturation remain unknown. Unfortunately, other well-described approaches based on neighbouring information could not be applied in this case since we found that genomic information from members of the Bacteroidales order is lacking compared with other taxonomic groups.\u003c/p\u003e \u003cp\u003eWhile the link between the observed antimicrobial activity and the identified BGCs could not be established, no additional BGCs were identified using common mining tools. Which may suggest a causal link between the observed halos and the identified peptides, in particular for the BGC1.The novel gene clusters found provide an entrance to discovering and characterising novel antimicrobial compounds from Bacteroidota. This will require further efforts in their isolation, or heterologous expression, purification and characterisation, which are future plans.\u003c/p\u003e \u003cp\u003eThe advent of new genome-mining tools[37,38,63,80,81] and the accessibility of genomic databases increased natural product discovery.[78,82\u0026ndash;84] However, this did not translate equally to Gram-negative bacteria, which remain still some years behind in terms of mining for natural products. In this work, automated search with genome-mining tools proved inaccurate and incomplete, and only together with manual search could interesting BGCs be identified. This highlights the need for comprehensive knowledge of bacteriocins from Gram-negative bacteria specifically. Furthermore, to gain more insights into the molecular mechanism and role of the peptides identified, ideally, a reliable and species-optimised genetic toolbox for genetic engineering of Bacteroidota should be established. While some progress is underway[85], a Bacteroidota's tailored genetic toolbox will immensely contribute to the fundamental understanding of their ecology in the gut as well as their role in the disease state.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank BEI Resources, NIAID, NIH for providing the bacterial strains listed in \u003cstrong\u003eTable 1\u003c/strong\u003e, obtained as part of the Human Microbiome Project.\u003c/p\u003e\n\u003cp\u003eThis work was supported by Marie Sklodowska-Curie Actions (MSCA) and Innovative Training Networks, H2020-MSCA-ITN-2018, grant number: 813781, \u0026ldquo;BestTreat.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials.\u003c/p\u003e\n\u003cp\u003eConflict of interests\u003c/p\u003e\n\u003cp\u003eThe authors confirm that no known competing financial interests or personal relationships influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eConceptualization: DGM, MVFC, OPK. 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Oral Microbiol. 35 (2020) 181\u0026ndash;191. https://doi.org/10.1111/omi.12304.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 3 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"84abed66-6af9-4677-bea0-3d9fef815432","identifier":"10.13039/100010665","name":"H2020 Marie Skłodowska-Curie Actions","awardNumber":"813781","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Groningen","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oral microbiota, gut microbiota, antimicrobial activity, Bacteroidota, genome mining","lastPublishedDoi":"10.21203/rs.3.rs-3875369/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3875369/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe oral and gut microbiota constitute vastly diverse and complex ecosystems. Their presence affects local and distal organs, thus having a major role in health and disease. Bacteria forming these complex communities display social behaviour and can positively or negatively impact their neighbours. While the potential for antimicrobial production of Gram-positive bacteria has been widely investigated, the research on Gram-negative bacteria is lagging behind, also because current bioinformatic tools appear to be suboptimal to detect antimicrobial clusters in these bacteria. The present study investigates the antimicrobial potential of the Gram-negative Bacteroidota phylum members from oral and gut bacterial microbiota. For this purpose, several Bacteroidota strains of oral and gut origin were tested against each other, and the genomes of bacterial strains displaying interesting antimicrobial activity were mined. Several biosynthetic gene clusters were detected, and the potential peptide sequences were identified. These putative peptides showed low sequence similarity to each other. Still, all contained a Gly-Gly motif, probably representing the processing site of the prepeptide, and they shared a similar N-terminal region reminiscent of the TIGR04149 protein family. However, the cluster architecture differed between the biosynthetic gene clusters, indicating they contain different posttranslational modifications (PTMs). These findings highlight the potential for novel antimicrobial discovery in Gram-negative bacteria relevant to the human microbiota and their ecology.\u003c/p\u003e","manuscriptTitle":"Antimicrobial activity screening of Bacteroidota and genome-based analysis of their antimicrobial biosynthetic potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 10:21:37","doi":"10.21203/rs.3.rs-3875369/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77c8f4f5-9e9e-4b94-9e40-d017df329cb3","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28217599,"name":"General Microbiology"},{"id":28217600,"name":"Bioinformatics"}],"tags":[],"updatedAt":"2024-02-06T10:21:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-06 10:21:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3875369","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3875369","identity":"rs-3875369","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}