pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida

pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida | 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 pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida Anna Czerwinski, Julia Löwenstrom, Sören Franzenburg, Espen Elias Groth, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5767962/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2025 Read the published version in BMC Microbiology → Version 1 posted 7 You are reading this latest preprint version Abstract Background The bacterial second messenger c-di-GMP is known to influence the formation of biofilms and thereby persistence of pathogenic and beneficial bacteria in hosts. A previous evolution experiment with Pseudomonas lurida MYb11, occasional symbiont of the nematode Caenorhabditis elegans , led to the emergence of host-specialized variants with elevated intracellular c-di-GMP. Thus far, the molecular underpinnings of c-di-GMP-mediated host specialization were unknown in this symbiosis. Therefore, the current study aimed at identifying candidate molecular processes by combining transcriptomic and functional genetic analyses. Results We found that MYb11 host specialists differentially expressed genes related to attachment, motility and biofilm production, including pelD from the pel gene cluster. pelD deletion resulted in reduced intra-host competitive fitness, lower bacterial numbers in C. elegans and loss of biofilm biomass. Conclusion Our results identify pelD as a previously unknown key modulator of beneficial symbiont-host associations that acts downstream of c-di-GMP. pelD Pseudomonas lurida Caenorhabditis elegans c-di-GMP biofilm symbiosis host-microbe interaction Figures Figure 1 Figure 2 Background The biology of microbe-host associations and thus the function of a microbiome has gained increasing interest in recent years ( 1 ). In general, microbes can benefit from the space and nutrients provided by the host while protecting it from pathogens or providing metabolites and nutrients ( 2 – 4 ). The molecular underpinnings of microbe-host interactions are important to understand how microbes can associate with a host and be part of a microbiome ( 5 ). Studying individual microbe-host interactions rather than complex microbiomes can help uncover and causally link these molecular details. A well-described example is the symbiosis between Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes ( 6 ). The squid recruits the bacterial symbionts from the environment by using a chitin gradient as a chemoattractant, subsequently allowing the bacteria to colonize the light organ of the bobtail squid by forming a biofilm. This symbiosis is beneficial for both the host and the microbe. V. fischeri provides bioluminescence, which is used by the nocturnally active squid for counterillumination, while the bacteria obtain amino acids from their host ( 7 – 9 ). Another example is Caenorhabditis elegans and its microbiome member Pseudomonas lurida MYb11, which can protect its host from infection with Bacillus thuringiensis while colonizing the gut and influencing early reproduction in the worm with its boom-and-bust life cycle ( 2 , 10 , 11 ). We are only beginning to understand how microbes associate with a particular host and the molecular requirements for a stable microbe-host association across different hosts ( 5 , 12 , 13 ). The persistence of microorganisms with or in a host is one of several prerequisites for the establishment of a stable microbe-host association ( 14 ). On a molecular level, 3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger involved in processes such as biofilm formation, surface attachment, virulence, motility, transition from motile to sessile organisms and cell cycle regulation ( 15 ). It contributes to bacterial adaptation to and their persistence in a new environment ( 16 , 17 ). The role of c-di-GMP-mediated adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung is an area of ongoing research and the adaptations described include excessive biofilm formation through alginate production, loss of twitching motility, resistance to antibiotics and reduced virulence ( 18 , 19 ). Given the importance of c-di-GMP for the transition from a mobile to a sessile lifestyle, its effects on biofilm formation and flagellar function are crucial ( 15 , 17 ). In c-di-GMP-dependent biofilm formation of P. aeruginosa , both the psl and pel gene clusters are important for initial and mature biofilm formation, while the alg operon is important for mature biofilm formation ( 16 ). PelD is encoded by the pel gene cluster and has been identified in Pseudomonads as a membrane-bound c-di-GMP receptor that is involved in the production of the Pel-polysaccharides ( 20 ). In addition, c-di-GMP can regulate the rotation of flagella, bacterial motility and surface attachment. Flagellar rotation responds to changing environments and generates regulatory feedback loops that are critical for the establishment of mature biofilms ( 17 ). Although there is increasing evidence for the importance of c-di-GMP in the formation of symbiosis ( 13 ), the role of c-di-GMP in beneficial symbioses with hosts is less well understood than in infections. Thus far, c-di-GMP levels have been shown to play an important role in the association of V. fischeri with the Hawaiian squid ( 21 ), Aeromonas veronii with the zebrafish ( 22 ), and in plant beneficial bacteria ( 23 ). Recently, we have shown that c-di-GMP is also a key factor in the host adaptation of Pseudomonas lurida strain MYb11 to C. elegans ( 24 ). Using an evolution experiment, we found that evolved wrinkly MYb11 isolates were host-specialized through increased short-term persistence and in vitro biofilm formation ( 24 ). Genome analysis of the host specialists revealed mutations in the wspE , wspF and rph genes, which regulate intracellular c-di-GMP levels. Elevated c-di-GMP levels were demonstrated to cause the increased persistence of MYb11 and other Pseudomonads from the C. elegans microbiome and environment in the host ( 24 ). Despite the importance of c-di-GMP levels for microbe-host association, it is currently unknown which downstream targets of c-di-GMP underlie the beneficial interactions upon association with C. elegans . Therefore, the aim of this study is to uncover the downstream targets of c-di-GMP in host-specialized MYb11 that enable adaptation to the native C. elegans strain MY316. We used comparative transcriptomics of ancestral and host-specialized MYb11 in different environments (liquid and solid media in vitro ) to identify differentially expressed genes, followed by gene set enrichment analysis. Based on the results, we genetically manipulated candidate genes in a representative host-specialist background of MYb11 to determine their effects on in vitro biofilm formation and persistence in the C. elegans MY316 host. We could show that pelD is required for the increased competitive fitness of the host-specialized MYb11, the total number of bacteria in the host, and also in vitro biofilm formation. We thus provide insights into the mechanism of c-di-GMP-driven adaptation of symbiotic bacteria to host association via a functional pel gene cluster. Materials and methods Overall strategy Since sequencing of bacterial transcripts during colonization of C. elegans is generally challenging, we decided to focus on the following two-step strategy: (i) Identification of MYb11-specific c-di-GMP downstream targets by RNA-Seq of three distinct mutants with upregulated c-di-GMP under two different media conditions (of these, the solid medium conditions are related to the environmental conditions of the previous evolution experiment; ( 24 ), and (ii) subsequent functional genetic analysis of selected MYb11 candidate genes for their ability to affect association of the bacterium with the C. elegans host. Host and bacterial strains The experiments were performed with the Pseudomonas lurida strain MYb11 and its natural host Caenorhabditis elegans strain MY316 ( 25 ). To prepare for the experiments with MY316, we thawed frozen worm strains (-70°C - -80°C, in either glycerol or DMSO stocks) and grew worms on nematode growth medium agar (NGM ( 26 )) inoculated with E. coli OP50 at an OD 600 of 3. A standard bleaching protocol was used to collect sterile and synchronized L1 larvae, which were then raised on appropriate bacterial lawns (20°C) to the L4 stage, as indicated in the individual sections. P. lurida strain MYb11 were isolated originally from MY316 ( 25 ). The host-specialized MYb11 isolates with mutations in wspE , wspF and rph were obtained from an earlier evolution experiment ( 24 ). Bacteria were cultured on tryptic soy agar (TSA; 20°C, 72 hours) and tryptic soy broth (TSB; 28°C, 150 rpm, overnight) unless otherwise stated. A list of the bacteria used and generated for genetic manipulation in this study can be found in Supplementary Table 20, Additional file 2. RNA sequencing In preparation for transcriptomic analysis of ancestral and wrinkly MYb11, RNA was isolated from liquid cultures (OD 600 = 1.8) and single colonies (20°C, 72h). For the liquid environment, a starter culture was prepared in 50 ml Falcon tubes. 10 ml TSB were inoculated with 100 µl of a MYb11 overnight culture and cultivated (28°C, 150 rpm) to OD 600 = 1.8. 1 ml of liquid cultures were centrifuged at 6000 rpm for 5 min, and pellets or colonies were resuspended in 800 µl TRI-zol™ reagent (ThermoFisher), frozen and stored (-80°C). RNA was isolated using the Direct-zol RNA Miniprep Kit (Zymo Research). NGS analyses were performed at the Competence Center for Genome Analysis (Kiel, Germany) using Illumina stranded total RNA library preparation and NovaSeq SP 2x50 bp sequencing. Transcriptome analyses To determine differentially expressed genes in the host-specialized P. lurida MYb11 compared to the ancestral MYb11, the RNA-Seq data were analyzed using high-performance computers at Kiel University Computer Center and R (version 4.1.1) with the following workflows and programs: Sequence quality control was assessed with FastQC (Babraham Institute) and MultiQC ( 27 ). Adapter trimming was achieved with Trimmomatic ( 28 ) in paired-end mode (version 0.39, phred33, adaptersUsed = TruSeq3-PE-2.fa, seedMismatches = 2, palindromeClipThreshold = 30, simpleClipThreshold = 10, minimumadapterlength = 2, lengthHeadcrop = 5, lengthMin = 36). Read mapping and read counting were performed with EDGE-pro set for paired-end reads ( 29 ), reference genome (NZ_CP023272.1) and files for Pseudomonas lurida MYb11 (Pseudomonas_lurida_MYb11_6243.csv.gz) and GO annotations ( Pseudomonas lurida MYb11 GO Term Annotations: gene_ontology_csv.csv) were obtained from Pseuomonas.com ( 30 ), mutations for the host specialists wspE , wspF and rph ( 24 ) were manually entered in the genome. Low read count filtering, data normalization, differential expression analysis with a negative binomial generalized linear model followed by a quasi-likelihood F-test and FDR correction for multiple testing, and Gene Set Enrichment Analysis (permutations = 1000, minimum gene set size = 2, and FDR correction for multiple testing) were performed using the R ( 31 ) packages edgeR version 4.2.1 ( 32 ) and clusterProfiler version 4.12.6 ( 33 ). KEGG Orthology was inferred using the KAAS online tool ( 34 ) (settings: GHOSTX, pep, all pseudomonas, BBH). Mutant generation A two-step allele replacement procedure based on previously described protocols ( 24 , 35 , 36 ) was used to delete candidate genes in the wspE host specialist background. In detail, ~ 700 bp long PCR amplicons surrounding each mutation were cloned into pUISacB, allowing sucrose selection. The constructs were transformed into competent E. coli cells and transferred to wspE host specialists by tri parental conjugation with an E. coli helper strain containing pRK2013 ( 37 ). Primers (Supplementary Table 21, Additional file 2) were designed using SnapGene software ( www.snapgene.com ) and NEBuilder v2.10.2 (New England Biolabs). Biofilm formation To determine the adherent biofilm biomass we used previously described protocols ( 24 , 38 ). In detail, the bacterial cultures were adjusted to OD 600 = 0.1 with M9 buffer and diluted 1:10 with TSB. Subsequently, 180 µl of the dilutions were transferred to a 96-well, flat-bottomed polystyrene microtiter plate. The plates were incubated for 48 hours at 20°C and 125 rpm. 200 µl of 0.01% crystal violet was added, incubated for 30 minutes at room temperature, washed four times with 300 µl ddH2O, 200 µl of acetic acid was added and incubated for 30 minutes at room temperature. The absorbance was measured at 590 nm, 550 nm and 530 nm. Bacterial persistence in worms We quantified the persistence in worms from the L4 stage onwards, as described before (see Early colonization, persistence and release in worms) ( 24 ). Bacterial lawns were prepared on NGM (125 µl, OD 600 = 2) from ancestral and mutated MYb11 (overnight cultures: 28°C, 150 rpm). For persistence assays, 40 synchronized worms were raised on the respective bacteria (from L1 to L4 stage, 20°C). The worms were collected with M9 buffer containing 0.025% Triton-100 and 25 mM of the paralyzing antihelminthic agent tetramisole. Worms were washed in buffer using a custom-made filter tip wash system ( 39 ) and then suspended in 200 µl of M9 and incubated for 1 hour (20°C), after which 100 µl of supernatant was collected. After homogenization with beads (1 mm zirconia), serial dilutions of worm suspension were plated and CFUs quantified. CFU/worm was calculated as the difference in CFU between worm and supernatant samples divided by the number of worms. Competitive fitness in MY316 host Competition experiments were performed as described for the persistence experiments and described before (see In vivo competition assays) ( 24 ). Co-inoculated bacteria were adjusted to OD 600 = 2 and mixed in equal amounts before seeding as lawns on NGM agar. A MYb11 labeled with dTomato ( 10 ) was used, which corresponds to the ancestral MYb11, as no differences were observed in short-term persistence (Fig. 2 ). The competitive index was calculated as the ratio of CFU/worm of the evolved or generated mutants to CFU/worm of the ancestor. Statistical analyses Prior to data analysis, the assumptions of parametric tests (normality, homogeneity of variances) were checked with Shapiro-Wilk and F-tests (Supplementary Tables 14–19, Additional file 2). If these were not met, non-parametric tests were applied. FDR correction was used for multiple testing. Box plots show the median (center line), the upper/lower quartiles (box boundaries) and the 1.5-fold interquartile ranges (whiskers). All analyses and plots were performed in R/RStudio ( 40 ) using the packages ggplot2 ( 41 ), VennDiagram ( 42 ), cowplot ( 43 ), plotly ( 44 ), and dplyr ( 45 ) as well as Inkscape. Results Host specialists show enriched expression of genes related to cell adhesion, flagellar function and biofilm formation In a previous study, we showed that c-di-GMP is a key factor for the adaptation of MYb11 to its host C. elegans MY316. Host adaptation was caused by mutations in the wspE , wspF , and rph , which all lead to elevated c-di-GMP levels ( 24 ). However, to date, the downstream targets of c-di-GMP in this adaptation remain unresolved. As a first step to identify potential downstream targets, we performed transcriptomic analyses of ancestral- versus the three host-specialized MYb11 mutants (i.e., with mutations in the genes wspE , wspF , and rph ) in different culture environments, including a solid environment on agar (i.e., tryptic soy agar, TSA) and a liquid medium (tryptic soy broth, TSB). A principal component analysis (PCA) of the mapped RNA-Seq counts per million (CPM) reads of ancestral and host-specialized MYb11 in the different environments showed that 72% (PC1) of the variation could be explained by the different culture environments, while 5.7% could be explained by the genomic background of the samples (PC3) (Fig. 1 a, Supplementary Figs. 1–3 for PC2 and PCAs for each environmental conditions and Supplementary Table 1, Additional files 1–2). In this PCA, the samples from solid medium show a more compact distribution than those from liquid medium. Considering that the PCA uses expression data from all genes for the four strains, this result suggests that there is overall less variation among strains under solid conditions. For the subsequent differential gene expression, samples from different culture environments were analyzed separately to focus on the difference between the ancestral and host-specialized MYb11 (Fig. 1 b; lists of differentially expressed genes (DEG), are given in Supplementary Tables 2–7, Additional file 2). The distribution of DEGs for the wspE mutant compared with the ancestor are illustrated in Fig. 1 c for the two media conditions separately. A few examples of significantly DEGs are highlighted, including those analyzed further through genetic manipulation below (Fig. 1 c). To characterize potential downstream functions of upregulated c-di-GMP, we then focused on the overlap of DEGs among the three mutants (850 in liquid medium, 104 DEGs on solid medium, Fig. 1 b) Supplementary Tables 8–9 in Additional file 2) and subjected these gene sets to a gene set enrichment analysis (GSEA) using gene ontology (GO) terms. The results reveal eight distinct significantly enriched GO terms under liquid conditions. Of these, the GO terms cell adhesion, pilus, flagellum-dependent cell motility, flagellum basal body, and motor activity are likely important for host specialization (Fig. 1 d, FDR < 0.05, Supplementary Table 10 in Additional file 2). Under solid conditions, only the GO term for alginic acid biosynthesis was significantly enriched. (Fig. 1 d, FDR < 0.05, Supplementary Table 11, Additional file 2). Overall, the transcriptome analysis of the host-specialized P. lurida MYb11 revealed that genes related to cell adhesion, such as the fimbrial genes, flagellar function and biofilm formation, such as the alg and pel gene clusters, are potential downstream targets of c-di-GMP. pelD , but none of the other tested candidate genes contributes to MYb11 competitive fitness and biofilm formation In a next step, we aimed to validate the role of the differentially expressed genes as downstream targets of c-di-GMP and therefore selected genes for genetic manipulation. For this, we focused on genes with (i) significant and high differential expression, and (ii) putative functions that match those identified in the GSEA (see red labelled genes in Fig. 1 c, Table 1 ). fimA and fimD were selected as genes involved in pilus formation and surface adhesion ( 46 ). Further, we selected azu , which is linked to cell copper homeostasis and type VI secretion ( 47 ). Notably, azu was found to be upregulated in P. aeruginosa from the lungs of patients with chronic cystic fibrosis ( 48 , 49 ). The flagellar motor switch genes fliM and fliN , which are described to be involved in flagellar rotation and surface recognition ( 17 ) were chosen for the investigation of flagellar function. As genes indicative of biofilm formation and direct regulatory interaction with c-di-GMP ( 50 ), we also selected algD, alg44 and pelD . Table 1 Candidate genes for genetic manipulation in wspE host specialist Name Gene product GO term Gene locus_tag Log2FC fimA type 1 fimbrial protein cell adhesion / pilus CLM75_RS17845 3.846 Azu azurin copper ion binding CLM75_RS02765 1.982 fliM flagellar motor switch protein FliM motor activity CLM75_RS19420 1.705 fliN flagellar motor switch protein FliN motor activity CLM75_RS19415 1.306 pelD sugar transporter - CLM75_RS01470 1.194 algD nucleotide sugar dehydrogenase alginic acid biosynthetic process CLM75_RS05035 1.163 fimD fimbrial protein pilus assembly CLM75_RS17855 1.132 alg44 alginate biosynthesis protein Alg44 cyclic-di-GMP binding CLM75_RS05020 0.639 Legend to the table: The candidate genes have significant and high differential expression (FDR < 0.05, log2FC ≥ ± 1), and/or putative functions consistent with the functions identified in the GSEA (Fig. 1 c, 1 d, Supplementary Tables 8–11, Additional file 2). NZ_CP023272.1 was used as the reference genome for read mapping and enrichment with GO terms (Supplementary Table 12, Additional file 2); the names correspond to the KEGG Orthology (Supplementary Table 13, Additional file 2). After selecting candidate genes for potential c-di-GMP downstream targets, we generated gene knockouts in the wspE host specialist background. We focused on this specific mutant background because it showed the most robust host-specialist phenotype in various host assays ( 24 ). We examined the competitive fitness of the generated mutants in competition with the ancestral MYb11 in the natural C. elegans MY316 host. Competitive fitness was performed as a short-term persistence assay and yielded data on colony-forming units (CFU) for the competing bacterial strains per worm host ( 24 ). We found that all ∆algD , ∆alg44 , ∆azu , ∆fimA , ∆fliM∆fliN and ∆fimD mutants maintained significantly higher competitive fitness compared to the ancestral MYb11 and thus did not lose the high competitiveness of the wspE -mutant host specialist (Fig. 2 a left panel, Supplementary Table 14, Additional file 2). Because the switch from a mobile to a sessile lifestyle mediated by c-di-GMP is often reflected by multiple changes in gene expression ( 51 ), we exemplarily tested whether the deletion of more than one gene was needed to reverse the host specialist phenotype. In detail, we generated ∆fimA∆fimD to test whether we could disrupt adhesion with multiple knockouts in the same pathway, ∆fliM∆fliN∆fimD to disrupt adhesion and different flagellar swimming modes, ∆pelD to disrupt Pel-polysaccharides as another upregulated biofilm gene cluster, and ∆pelD∆fimD to disrupt Pel-polysaccharide production and adhesion. Of these mutants, ∆pelD and ∆pelD∆fimD showed the lower ancestral competitive fitness and thus lost one of the important characteristics of the host specialist wspE mutant (Fig. 2 a, right panel, Supplementary Table 14, Additional file 2). In a next step, we investigated how the mutations affect the fitness of the population within the host (total number of CFU/worm as CFU/worm mutant + ancestor). The mutants ∆algD , Δalg44 , ∆fliM∆fliN , ΔfimD , ∆pelD and ∆pelD∆fimD lost the significantly increased CFU/worm number in competition compared to the ancestral MYb11 (Fig. 2 b). Notably, only the competitions with ∆pelD and ∆pelD∆fimD mutants showed a significantly reduced CFU/worm number compared to wspE host specialist (Fig. 2 b, Supplementary Tables 15–16, Additional file 2). Similar trends, although not significant, were observed for mono-colonization of ∆pelD and ∆pelD∆fimD mutants compared to wspE host specialists (Supplementary Fig. 4, Supplementary Tables 17–18, Additional files 1–2). In addition, we analyzed the potential of the mutants to adhere to surfaces by forming biofilms using a crystal violet microtiter plate adherence assay. We included E. coli OP50 as a negative control for biofilm formation ( 52 ). The increased adherent biofilm biomass was lost in ∆algD , Δalg44 , Δazu , ∆fliM∆fliN , ∆pelD , ΔfimAΔfimD , ∆fliM∆fliN∆fimD and ∆pelD∆fimD mutants compared to ancestral P. lurida MYb11. However, a significant decrease was only observed for ∆pelD and ∆pelD∆fimD as compared to the ancestral MYb11 and the wspE host specialist (Fig. 2 c, Supplementary Table 19, Additional file 2). In summary, the ∆pelD and ∆pelD∆fimD mutants in the wspE host specialist background have lost two important features of the host-specialized MYb11, namely increased host competitive fitness in the host and increased biomass of the adherent biofilm. Although other genes identified in the transcriptome analyses (Fig. 1 ) have already been linked to c-di-GMP-mediated shifts in Pseudomonads ( 17 , 46 – 50 ), we were only able to causally link pelD to the observed adaptive traits of the host-specialized P. lurida MYb11 (Fig. 2 ). Discussion In this study, we characterized possible downstream targets of c-di-GMP-mediated adaptation of host-specialized P. lurida MYb11. Our transcriptome analysis showed that the three different host specialist mutants varied from the ancestral MYb11 strain in gene expression for functions related to cell adhesion, flagellar function and biofilm formation. Our subsequent functional genetic analysis revealed that only the deletion of pelD was able to reverse the host specialist phenotype in the tested traits: competitive fitness in the host, total cell number in the host, and biofilm formation. Thus, our results emphasize the importance of pelD for the persistence and the biofilm phenotype of MYb11 host specialists, while other functions discovered in the transcriptome analyses may only play a minor role, at least at the level of a single gene. PelD has been shown to post-transcriptionally regulate the production of Pel-polysaccharides in P. aeruginosa in a c-di-GMP-dependent manner ( 50 , 53 ). c-di-GMP binds to the degenerate GGDEF domain of the inner membrane protein PelD, leading to a conformational change and thus enabling the biosynthesis of Pel-polysaccharides ( 50 , 53 ). Functional predictions of Interpro ( 54 ) retrieved from Pseudomonas.com ( 30 ) indicate that this degenerate GGDEF domain is also part of the PelD of P. lurida MYb11. Furthermore, it was previously shown that pelD is absolutely required for the production of Pel polysaccharides in P. aeruginosa ( 55 ), suggesting that deletion of pelD in P. lurida MYb11 also disrupts the production of Pel polysaccharides. It follows that the higher competitive fitness of host specialists, and the enhanced biofilm formation mediated by c-di-GMP are specifically and highly dependent on a functioning pel gene cluster and Pel-polysaccharides. Moreover, MYb11 is a Pseudomonad that harbors the major exopolysaccharide gene clusters alg , wss , psl and pel ( 56 ), but our data suggest that none of the remaining gene clusters can compensate for the production of Pel-polysaccharides, indicating that Pel-polysaccharides are the major component of the adherent biofilm of MYb11. This study focused on the downstream targets of c-di-GMP that are important for host association. However, the host-specialized P. lurida MYb11 evolved in a life cycle consisting of a host-associated and a free-living phase. The lack of an effect on the host association of other upregulated genes in the host-specialized MYb11 could have several reasons: Firstly, the lack of effect could simply be explained by genomic redundancy, i.e. genes in the tested signaling pathway, but also in the entire genome, could take over the lost function. This could be the case, for example, for genes involved in the production of fimbriae. In the MYb11 genome of P. lurida , other genes encoding fimbrial proteins such as fimA and fimD are present (CLM75_RS01985, CLM75_RS01970 and CLM75_RS17200, Supplementary Tables 12–13, Additional file 2). On the other hand, the upregulated genes might play a role for survival in a free-living environment and be less important inside the host. For example, genes related to alginate production have been shown to be important for the adaptation and survival of P. fluorescens PF0-1 in dehydrated soil ( 57 ). In another soil bacterium, P. putida , the regulation of flagellar rotation and thus swimming mode has been shown to be important for motility in different liquid and solid environments ( 58 ). In contrast, azurin is used by Pseudomonads to maintain copper-ion homeostasis in combination with TonB-dependent receptors and type VI secretion systems and therefore may play a role in nutrient acquisition in a free-living environment ( 47 ). A detailed knowledge of how microbes adapt to the host association and persist in a host is crucial for full appreciation of the formation and functioning of host-microbiome interactions. Investigating the molecular basis of these adaptations in a single bacterial-host association will help us to identify conserved mechanisms as well as species-specific differences between microbes. In our work, we use C. elegans MY316 and its symbiont P. lurida MYb11 to study bacterial adaptation to a host. c-di-GMP and Pel-polysaccharides have been shown to be involved in host association, especially of pathogenic bacteria ( 59 ). One example is the association of P. aeruginosa with cystic fibrosis lung, where Pel-polysaccharides contribute to the biofilm matrix that allows the bacteria to persist in the lung and protect it from host immune defenses and antibiotics ( 60 ). To our knowledge, Pel-polysaccharides have not yet been shown to be involved in beneficial host-microbe associations. Thus, it is conceivable that many of the mechanisms previously reported to shape the much more intensively studied interactions between host and pathogens are actually involved in any kind of symbiotic interaction, ranging from mutualistic over commensal to pathogenic associations. Overall, the current study extends our previous work ( 24 ) by providing new insights into the mechanisms of c-di-GMP-driven host adaptation in symbioses beyond infection and demonstrates the specific role of pelD that acts downstream of c-di-GMP to mediate the competitive fitness of Pseudomonas lurida MYb11 within its nematode host Caenorhabditis elegans MY316. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The transcriptome sequence data is available from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) under the following accession number: GSE288391. Other datasets generated and analyzed during the current study are available from our pelD_c-di-GMP_host_specialization Github repository: https://github.com/evoecogen/pelD_c-di-GMP_host_specialization.git Competing interests The authors declare that they have no competing interests Funding We are grateful for funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Project-ID 261376515 – SFB 1182, Projects A4.3 and B4.3 (NO, HS); the DFG Clinician Scientist Program in Evolutionary Medicine „CSEM“ project 413490537 (EEG), the DFG Research Infrastructure NGS_CC project 407495230 (SF) as part of the Next Generation Sequencing Competence Network project 423957469; the International Max-Planck Research School for Evolutionary Biology (NO, AC); and the Max-Planck Society (Fellowship to HS). Authors' contributions A.C., N.O. and H.S. conceptualized the project. A.C., N.O. and E.E.G. developed the methodology. A.C., N.O., J.L. and S.F. conducted investigations. A.C., N.O. and J.L. analyzed data. A.C., N.O. and H.S. contributed to the writing of the manuscript. N.O. and H.S. supervised the project. Acknowledgments We thank P. Rainey, D. Rogers, J. Summers (Max-Planck Institute for Evolutionary Biology, Ploen, Germany) for providing bacterial strains and plasmids, and advice on allelic exchange; J. Zimmermann, Daniel Schütz (Schulenburg group, University of Kiel, Germany) for bioinformatic support; J. Hofmann, H. Griem-Krey, L. Bluhm, L. Rheindorf, K. Flinder and N. Steinbach (all Schulenburg group, University of Kiel, Germany) for lab support; the Kiel BiMo/LMB for access to their core facilities; S. Koehler (Schulenburg group, University of Kiel, Germany) and H. Sondermann (Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany) for advice on the manuscript; the Schulenburg lab for project feedback. References Bosch TCG, Blaser MJ, Ruby E, McFall-Ngai M. A new lexicon in the age of microbiome research. Philos Trans R Soc Lond B Biol Sci. 2024 May 6;379(1901):20230060. Kissoyan KAB, Drechsler M, Stange EL, Zimmermann J, Kaleta C, Bode HB, et al. Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr Biol. 2019 Mar;29(6):1030-1037.e5. Engel P, Moran NA. The gut microbiota of insects - diversity in structure and function. FEMS Microbiol Rev. 2013 Sep;37(5):699–735. Ramírez-Pérez O, Cruz-Ramón V, Chinchilla-López P, Méndez-Sánchez N. The Role of the Gut Microbiota in Bile Acid Metabolism. Ann Hepatol. 2017 Nov;16(Suppl. 1: s3-105.):s15–20. Singh A, Luallen RJ. Understanding the factors regulating host-microbiome interactions using Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 2024 May 6;379(1901):20230059. McFall-Ngai M. Host-Microbe Symbiosis: The Squid-Vibrio Association—A Naturally Occurring, Experimental Model of Animal/Bacterial Partnerships. In: Huffnagle GB, Noverr MC, editors. GI Microbiota and Regulation of the Immune System [Internet]. New York, NY: Springer; 2008 [cited 2024 Nov 19]. p. 102–12. Available from: https://doi.org/10.1007/978-0-387-09550-9_9 Nyholm SV, McFall-Ngai MJ. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat Rev Microbiol. 2021 Oct;19(10):666–79. Visick KL, Stabb EV, Ruby EG. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat Rev Microbiol. 2021 Oct;19(10):654–65. Kremer N, Philipp EER, Carpentier MC, Brennan CA, Kraemer L, Altura MA, et al. Initial Symbiont Contact Orchestrates Host-Organ-wide Transcriptional Changes that Prime Tissue Colonization. Cell Host Microbe. 2013 Aug 14;14(2):183–94. Kissoyan KAB, Peters L, Giez C, Michels J, Pees B, Hamerich IK, et al. Exploring Effects of C. elegans Protective Natural Microbiota on Host Physiology. Front Cell Infect Microbiol. 2022 Feb 14;12:775728. Griem-Krey H, Petersen C, Hamerich IK, Schulenburg H. The intricate triangular interaction between protective microbe, pathogen and host determines fitness of the metaorganism. Proc Biol Sci. 2023 Dec 6;290(2012):20232193. Obeng N, Bansept F, Sieber M, Traulsen A, Schulenburg H. Evolution of Microbiota–Host Associations: The Microbe’s Perspective. Trends Microbiol. 2021 Sep 1;29(9):779–87. Isenberg RY, Mandel MJ. Cyclic Diguanylate in the Wild: Roles During Plant and Animal Colonization. Annu Rev Microbiol [Internet]. 2024 Sep 13 [cited 2024 Sep 24]; Available from: https://www.annualreviews.org/content/journals/10.1146/annurev-micro-041522-101729 Robinson CD, Bohannan BJ, Britton RA. Scales of persistence: transmission and the microbiome. Curr Opin Microbiol. 2019 Aug 1;50:42–9. Römling U, Galperin MY, Gomelsky M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev MMBR. 2013 Mar;77(1):1–52. Ha DG, O’Toole GA. c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.MB-0003–2014. Laventie BJ, Jenal U. Surface Sensing and Adaptation in Bacteria. Annu Rev Microbiol. 2020 Sep 8;74:735–60. Koska M, Kordes A, Erdmann J, Willger SD, Thöming JG, Bähre H, et al. Distinct Long- and Short-Term Adaptive Mechanisms in Pseudomonas aeruginosa. Microbiol Spectr. 2022 Dec 21;10(6):e0304322. Ruhluel D, Fisher L, Barton TE, Leighton H, Kumar S, Amores Morillo P, et al. Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments. ISME J. 2024 Jan 8;18(1):wrae065. Franklin M, Nivens D, Weadge J, Howell P. Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl. Front Microbiol [Internet]. 2011 [cited 2023 Dec 21];2. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2011.00167 Isenberg RY, Christensen DG, Visick KL, Mandel MJ. High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization. mBio. 2022 Aug 30;13(4):e0167122. Robinson CD, Sweeney EG, Ngo J, Ma E, Perkins A, Smith TJ, et al. Host-emitted amino acid cues regulate bacterial chemokinesis to enhance colonization. Cell Host Microbe. 2021 Aug 11;29(8):1221-1234.e8. Huang W, Wang D, Zhang XX, Zhao M, Sun L, Zhou Y, et al. Regulatory roles of the second messenger c-di-GMP in beneficial plant-bacteria interactions. Microbiol Res. 2024 Aug;285:127748. Obeng N, Czerwinski A, Schütz D, Michels J, Leipert J, Bansept F, et al. Bacterial c-di-GMP has a key role in establishing host–microbe symbiosis. Nat Microbiol. 2023 Aug 31;1–11. Dirksen P, Marsh SA, Braker I, Heitland N, Wagner S, Nakad R, et al. The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model. BMC Biol. 2016 Dec;14(1):38. Stiernagle T. Maintenance of C. elegans. WormBook [Internet]. 2006 [cited 2020 Dec 20]; Available from: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047–8. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014 Aug 1;30(15):2114–20. Magoc T, Wood D, Salzberg SL. EDGE-pro: Estimated Degree of Gene Expression in Prokaryotic Genomes. Evol Bioinforma Online. 2013 Mar 10;9:127–36. Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FSL. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 2016 Jan 4;44(D1):D646-653. R Core Team. R: A Language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2024. Available from: https://www.R-project.org/ Chen Y, Chen L, Lun ATL, Baldoni PL, Smyth GK. edgeR 4.0: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets [Internet]. Bioinformatics; 2024 [cited 2024 Oct 15]. Available from: http://biorxiv.org/lookup/doi/10.1101/2024.01.21.576131 Xu S, Hu E, Cai Y, Xie Z, Luo X, Zhan L, et al. Using clusterProfiler to characterize multiomics data. Nat Protoc. 2024 Jul 17;1–29. Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007 Jul;35(Web Server issue):W182-185. Bantinaki E, Kassen R, Knight CG, Robinson Z, Spiers AJ, Rainey PB. Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens . III. Mutational Origins of Wrinkly Spreader Diversity. Genetics. 2007 May;176(1):441–53. Hmelo LR, Borlee BR, Almblad H, Love ME, Randall TE, Tseng BS, et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc. 2015 Nov;10(11):1820–41. Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci. 1979 Apr 1;76(4):1648–52. O’Toole GA. Microtiter Dish Biofilm Formation Assay. J Vis Exp. 2011 Jan 30;(47):2437. Papkou A, Guzella T, Yang W, Koepper S, Pees B, Schalkowski R, et al. The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution. Proc Natl Acad Sci U S A. 2019 Jan 15;116(3):923–8. Posit team. RStudio: Integrated Development Environment for R [Internet]. Boston, MA: Posit Software, PBC; 2024. Available from: http://www.posit.co/ Wickham H. ggplot2: Elegant Graphics for Data Analysis. 2nd ed. 2016. Cham: Springer International Publishing : Imprint: Springer; 2016. 1 p. (Use R!). Hanbo Chen. VennDiagram: Generate High-Resolution Venn and Euler Plots [Internet]. 2011 [cited 2024 Oct 15]. p. 1.7.3. Available from: https://CRAN.R-project.org/package=VennDiagram Wilke CO. cowplot: Streamlined Plot Theme and Plot Annotations for “ggplot2” [Internet]. 2015 [cited 2024 Oct 15]. p. 1.1.3. Available from: https://CRAN.R-project.org/package=cowplot Li R, Bilal U. Interactive Web-Based Data Visualization with R, Plotly, and Shiny (Carson Sievert). Biometrics. 2021 Jun 1;77(2):776–7. Wickham H, François R, Henry L, Müller K, Vaughan D, Software P, et al. dplyr: A Grammar of Data Manipulation [Internet]. 2023 [cited 2024 Oct 15]. Available from: https://cran.r-project.org/web/packages/dplyr/index.html Böhning J, Dobbelstein AW, Sulkowski N, Eilers K, von Kügelgen A, Tarafder AK, et al. Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa. PLOS Pathog. 2023 Apr 14;19(4):e1011177. Han Y, Wang T, Chen G, Pu Q, Liu Q, Zhang Y, et al. A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition. PLoS Pathog. 2019 Dec 2;15(12):e1008198. Wood SJ, Goldufsky JW, Seu MY, Dorafshar AH, Shafikhani SH. Pseudomonas aeruginosa Cytotoxins: Mechanisms of Cytotoxicity and Impact on Inflammatory Responses. Cells. 2023 Jan;12(1):195. Hogardt M, Heesemann J. Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung. Int J Med Microbiol. 2010 Dec 1;300(8):557–62. Gheorghita AA, Wozniak DJ, Parsek MR, Howell PL. Pseudomonas aeruginosa biofilm exopolysaccharides: assembly, function, and degradation. FEMS Microbiol Rev. 2023 Nov 1;47(6):fuad060. Valentini M, Filloux A. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria. J Biol Chem. 2016 Jun 10;291(24):12547–55. Arata Y, Oshima T, Ikeda Y, Kimura H, Sako Y. OP50, a bacterial strain conventionally used as food for laboratory maintenance of C. elegans, is a biofilm formation defective mutant. MicroPublication Biol. 2020:10.17912/micropub.biology.000216. Whitney JC, Colvin KM, Marmont LS, Robinson H, Parsek MR, Howell PL. Structure of the Cytoplasmic Region of PelD, a Degenerate Diguanylate Cyclase Receptor That Regulates Exopolysaccharide Production in Pseudomonas aeruginosa. J Biol Chem. 2012 Jul 6;287(28):23582–93. Paysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, et al. InterPro in 2022. Nucleic Acids Res. 2023 Jan 6;51(D1):D418–27. Whitfield GB, Marmont LS, Ostaszewski A, Rich JD, Whitney JC, Parsek MR, et al. Pel Polysaccharide Biosynthesis Requires an Inner Membrane Complex Comprised of PelD, PelE, PelF, and PelG. J Bacteriol. 2020 Mar 26;202(8):e00684-19. Heredia-Ponce Z, de Vicente A, Cazorla FM, Gutiérrez-Barranquero JA. Beyond the Wall: Exopolysaccharides in the Biofilm Lifestyle of Pathogenic and Beneficial Plant-Associated Pseudomonas. Microorganisms. 2021 Feb 21;9(2):445. Marshall DC, Arruda BE, Silby MW. Alginate genes are required for optimal soil colonization and persistence by Pseudomonas fluorescens Pf0-1. Access Microbiol. 2019 May 3;1(3):e000021. Pfeifer V, Beier S, Alirezaeizanjani Z, Beta C. Role of the Two Flagellar Stators in Swimming Motility of Pseudomonas putida. mBio. 13(6):e02182-22. Valentini M, Filloux A. Multiple Roles of c-di-GMP Signaling in Bacterial Pathogenesis. Annu Rev Microbiol. 2019 Sep 8;73:387–406. Chung J, Eisha S, Park S, Morris AJ, Martin I. How Three Self-Secreted Biofilm Exopolysaccharides of Pseudomonas aeruginosa, Psl, Pel, and Alginate, Can Each Be Exploited for Antibiotic Adjuvant Effects in Cystic Fibrosis Lung Infection. Int J Mol Sci. 2023 May 13;24(10):8709. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1Czerwinskietal20241215.pdf Additionalfile2Czerwinskietal20250314.xlsx Additionalinformation.docx Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Accepted 01 Apr, 2025 Reviews received at journal 24 Mar, 2025 Reviewers agreed at journal 22 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Submission checks completed at journal 19 Mar, 2025 First submitted to journal 19 Mar, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5767962","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431401825,"identity":"09851e43-c76b-4b88-87eb-08f1514d8abf","order_by":0,"name":"Anna Czerwinski","email":"","orcid":"","institution":"University of Kiel","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Czerwinski","suffix":""},{"id":431401826,"identity":"018a241b-9047-4bed-9d60-641281c847b5","order_by":1,"name":"Julia Löwenstrom","email":"","orcid":"","institution":"University of Kiel","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Löwenstrom","suffix":""},{"id":431401827,"identity":"b9b69647-5b3a-4ceb-bc41-42df8cedf52e","order_by":2,"name":"Sören Franzenburg","email":"","orcid":"","institution":"University of Kiel","correspondingAuthor":false,"prefix":"","firstName":"Sören","middleName":"","lastName":"Franzenburg","suffix":""},{"id":431401828,"identity":"38bac594-74d3-43e1-96b4-e64e1f7dfac9","order_by":3,"name":"Espen Elias Groth","email":"","orcid":"","institution":"LungenClinic Grosshansdorf","correspondingAuthor":false,"prefix":"","firstName":"Espen","middleName":"Elias","lastName":"Groth","suffix":""},{"id":431401829,"identity":"f5d2ec55-374a-4797-b239-0968b7b328db","order_by":4,"name":"Nancy Obeng","email":"","orcid":"","institution":"University of Kiel","correspondingAuthor":false,"prefix":"","firstName":"Nancy","middleName":"","lastName":"Obeng","suffix":""},{"id":431401830,"identity":"88323f96-3817-45ce-8fdd-fcdae6e912b3","order_by":5,"name":"Hinrich Schulenburg","email":"data:image/png;base64,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","orcid":"","institution":"University of Kiel","correspondingAuthor":true,"prefix":"","firstName":"Hinrich","middleName":"","lastName":"Schulenburg","suffix":""}],"badges":[],"createdAt":"2025-01-05 13:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5767962/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5767962/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-03945-1","type":"published","date":"2025-04-16T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78942151,"identity":"fbc1ba6b-9ae6-463b-9a04-096027c347b9","added_by":"auto","created_at":"2025-03-21 07:04:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1481193,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome differences between host-specialized and ancestral \u003cem\u003eP. lurida\u003c/em\u003e MYb11.\u003c/p\u003e\n\u003cp\u003ea Principal component analysis (PCA) of log counts per million (CPM) reads of MYb11 and the evolved wrinkly \u003cem\u003ewspF\u003c/em\u003e, \u003cem\u003ewspE\u003c/em\u003e and\u003cem\u003e rph\u003c/em\u003emutants in liquid and solid environments (both tryptic soy based; Supplementary Table 1, Additional file 2). b Shared gene expression signature of the three host specialists. Venn diagrams show the significantly and differentially expressed genes of the evolved host-specialist mutants compared to the ancestral MYb11 (significantly DEG: FDR \u0026lt; 0.05, Supplementary Tables 8-9, Additional file 2). c Volcano diagram showing the DEG of the \u003cem\u003ewspE \u003c/em\u003ehost specialist (Supplementary Tables 2 \u0026amp; 5, Additional file 2). The cut-off value for the log2 change was set to -1 and 1 (blue and red dashed line), the cut-off value for the p-value to 0.05 (gray dashed line). The candidate genes selected for genetic manipulation are highlighted in red. d GSEA based on GO terms of the significantly DEG overlap of the host specialists (FDR \u0026lt; 0.05, Supplementary Tables 10-11, Additional file 2). RNA-Seq was performed with 5 independent biological replicates for each strain. Green: liquid environment, brown: solid environment. *:Indicates names annotated according to KEGG Orthology.\u003c/p\u003e","description":"","filename":"Czerwinskietal20251403Fig.1wb.png","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/4ae261a5cd7286a372cafc78.png"},{"id":78941832,"identity":"c3f75451-0c24-47d6-a212-af4dc56c9815","added_by":"auto","created_at":"2025-03-21 06:56:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":621961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epelD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ereduces bacterial fitness in the host and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eCompetitive fitness of MYb11 mutants in the \u003cem\u003eC. elegans\u003c/em\u003e MY316 host (CFU/worm compared to dTomato-labeled ancestor, Supplementary Table 14, Additional file 2).\u003cstrong\u003e b\u003c/strong\u003e Total CFU/per worm during short-term persistence competition in \u003cem\u003eC. elegans\u003c/em\u003e MY316 (Supplementary Tables 15-16, Additional file 2). \u003cstrong\u003ec\u003c/strong\u003e Biomass of the attached biofilm, measured as crystal violet absorbance at 550 nm (Supplementary Table 19, Additional file 2). The black dashed line and the p-values at the bottom of each graph refer to comparisons with the ancestral MYb11, the red dashed line and the p-values at the top refer to the \u003cem\u003ewspE \u003c/em\u003ehost specialist. Ancestral MYb11, evolved \u003cem\u003ewspE- \u003c/em\u003eand generated knockout mutants with \u003cem\u003ewspE\u003c/em\u003e or \u003cem\u003ewspE∆fimD\u003c/em\u003ebackground (3 \u0026lt; n \u0026lt; 5 replicates per strain). Statistical significance was determined using a t-test with equal variances, a Mann-Whitney U-test or a Welch's t-test with FDR corrections for multiple testing, depending on whether the parametric assumptions were met (Supplementary Tables 14-19, Additional file 2).\u003c/p\u003e","description":"","filename":"Czerwinskietal20250311Fig.2wb.png","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/37da4d9df90e0dc0946a81da.png"},{"id":81050603,"identity":"79cc87dd-d8e0-48ab-bf61-a0d8b932f8e6","added_by":"auto","created_at":"2025-04-21 16:00:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2509086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/18aefd21-5274-4802-bed0-44421af44598.pdf"},{"id":78942152,"identity":"bf0dfbf3-7164-4067-8997-71cd87fb2b3b","added_by":"auto","created_at":"2025-03-21 07:04:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":521386,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1Czerwinskietal20241215.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/403b710aba8a284de73216f2.pdf"},{"id":78941847,"identity":"96ae133b-9aec-489b-abd6-879b5f9dbe4d","added_by":"auto","created_at":"2025-03-21 06:56:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7378942,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2Czerwinskietal20250314.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/fcdb20a74bc62e6abfdeaa23.xlsx"},{"id":78941829,"identity":"cfd565b5-7436-4ae0-8055-ff56c5003fdb","added_by":"auto","created_at":"2025-03-21 06:56:19","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15512,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5767962/v1/96552468cc82bf51f2e0942f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida","fulltext":[{"header":"Background","content":"\u003cp\u003eThe biology of microbe-host associations and thus the function of a microbiome has gained increasing interest in recent years (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In general, microbes can benefit from the space and nutrients provided by the host while protecting it from pathogens or providing metabolites and nutrients (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The molecular underpinnings of microbe-host interactions are important to understand how microbes can associate with a host and be part of a microbiome (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Studying individual microbe-host interactions rather than complex microbiomes can help uncover and causally link these molecular details. A well-described example is the symbiosis between \u003cem\u003eVibrio fischeri\u003c/em\u003e and the Hawaiian bobtail squid \u003cem\u003eEuprymna scolopes\u003c/em\u003e (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The squid recruits the bacterial symbionts from the environment by using a chitin gradient as a chemoattractant, subsequently allowing the bacteria to colonize the light organ of the bobtail squid by forming a biofilm. This symbiosis is beneficial for both the host and the microbe. \u003cem\u003eV. fischeri\u003c/em\u003e provides bioluminescence, which is used by the nocturnally active squid for counterillumination, while the bacteria obtain amino acids from their host (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Another example is \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e and its microbiome member \u003cem\u003ePseudomonas lurida\u003c/em\u003e MYb11, which can protect its host from infection with \u003cem\u003eBacillus thuringiensis\u003c/em\u003e while colonizing the gut and influencing early reproduction in the worm with its boom-and-bust life cycle (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). We are only beginning to understand how microbes associate with a particular host and the molecular requirements for a stable microbe-host association across different hosts (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe persistence of microorganisms with or in a host is one of several prerequisites for the establishment of a stable microbe-host association (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). On a molecular level, 3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger involved in processes such as biofilm formation, surface attachment, virulence, motility, transition from motile to sessile organisms and cell cycle regulation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It contributes to bacterial adaptation to and their persistence in a new environment (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The role of c-di-GMP-mediated adaptation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e to the cystic fibrosis lung is an area of ongoing research and the adaptations described include excessive biofilm formation through alginate production, loss of twitching motility, resistance to antibiotics and reduced virulence (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Given the importance of c-di-GMP for the transition from a mobile to a sessile lifestyle, its effects on biofilm formation and flagellar function are crucial (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In c-di-GMP-dependent biofilm formation of \u003cem\u003eP. aeruginosa\u003c/em\u003e, both the \u003cem\u003epsl\u003c/em\u003e and \u003cem\u003epel\u003c/em\u003e gene clusters are important for initial and mature biofilm formation, while the \u003cem\u003ealg\u003c/em\u003e operon is important for mature biofilm formation (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). PelD is encoded by the \u003cem\u003epel\u003c/em\u003e gene cluster and has been identified in Pseudomonads as a membrane-bound c-di-GMP receptor that is involved in the production of the Pel-polysaccharides (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In addition, c-di-GMP can regulate the rotation of flagella, bacterial motility and surface attachment. Flagellar rotation responds to changing environments and generates regulatory feedback loops that are critical for the establishment of mature biofilms (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough there is increasing evidence for the importance of c-di-GMP in the formation of symbiosis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), the role of c-di-GMP in beneficial symbioses with hosts is less well understood than in infections. Thus far, c-di-GMP levels have been shown to play an important role in the association of \u003cem\u003eV. fischeri\u003c/em\u003e with the Hawaiian squid (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), \u003cem\u003eAeromonas veronii\u003c/em\u003e with the zebrafish (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and in plant beneficial bacteria (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Recently, we have shown that c-di-GMP is also a key factor in the host adaptation of \u003cem\u003ePseudomonas lurida\u003c/em\u003e strain MYb11 to \u003cem\u003eC. elegans\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Using an evolution experiment, we found that evolved wrinkly MYb11 isolates were host-specialized through increased short-term persistence and \u003cem\u003ein vitro\u003c/em\u003e biofilm formation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Genome analysis of the host specialists revealed mutations in the \u003cem\u003ewspE\u003c/em\u003e, \u003cem\u003ewspF\u003c/em\u003e and \u003cem\u003erph\u003c/em\u003e genes, which regulate intracellular c-di-GMP levels. Elevated c-di-GMP levels were demonstrated to cause the increased persistence of MYb11 and other Pseudomonads from the \u003cem\u003eC. elegans\u003c/em\u003e microbiome and environment in the host (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Despite the importance of c-di-GMP levels for microbe-host association, it is currently unknown which downstream targets of c-di-GMP underlie the beneficial interactions upon association with \u003cem\u003eC. elegans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study is to uncover the downstream targets of c-di-GMP in host-specialized MYb11 that enable adaptation to the native \u003cem\u003eC. elegans\u003c/em\u003e strain MY316. We used comparative transcriptomics of ancestral and host-specialized MYb11 in different environments (liquid and solid media \u003cem\u003ein vitro\u003c/em\u003e) to identify differentially expressed genes, followed by gene set enrichment analysis. Based on the results, we genetically manipulated candidate genes in a representative host-specialist background of MYb11 to determine their effects on \u003cem\u003ein vitro\u003c/em\u003e biofilm formation and persistence in the \u003cem\u003eC. elegans\u003c/em\u003e MY316 host. We could show that \u003cem\u003epelD\u003c/em\u003e is required for the increased competitive fitness of the host-specialized MYb11, the total number of bacteria in the host, and also \u003cem\u003ein vitro\u003c/em\u003e biofilm formation. We thus provide insights into the mechanism of c-di-GMP-driven adaptation of symbiotic bacteria to host association via a functional \u003cem\u003epel\u003c/em\u003e gene cluster.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOverall strategy\u003c/h2\u003e \u003cp\u003eSince sequencing of bacterial transcripts during colonization of \u003cem\u003eC. elegans\u003c/em\u003e is generally challenging, we decided to focus on the following two-step strategy: (i) Identification of MYb11-specific c-di-GMP downstream targets by RNA-Seq of three distinct mutants with upregulated c-di-GMP under two different media conditions (of these, the solid medium conditions are related to the environmental conditions of the previous evolution experiment; (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and (ii) subsequent functional genetic analysis of selected MYb11 candidate genes for their ability to affect association of the bacterium with the \u003cem\u003eC. elegans\u003c/em\u003e host.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHost and bacterial strains\u003c/h3\u003e\n\u003cp\u003eThe experiments were performed with the \u003cem\u003ePseudomonas lurida\u003c/em\u003e strain MYb11 and its natural host \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e strain MY316 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). To prepare for the experiments with MY316, we thawed frozen worm strains (-70\u0026deg;C - -80\u0026deg;C, in either glycerol or DMSO stocks) and grew worms on nematode growth medium agar (NGM (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)) inoculated with \u003cem\u003eE. coli\u003c/em\u003e OP50 at an OD\u003csub\u003e600\u003c/sub\u003e of 3. A standard bleaching protocol was used to collect sterile and synchronized L1 larvae, which were then raised on appropriate bacterial lawns (20\u0026deg;C) to the L4 stage, as indicated in the individual sections. \u003cem\u003eP. lurida\u003c/em\u003e strain MYb11 were isolated originally from MY316 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The host-specialized MYb11 isolates with mutations in \u003cem\u003ewspE\u003c/em\u003e, \u003cem\u003ewspF\u003c/em\u003e and \u003cem\u003erph\u003c/em\u003e were obtained from an earlier evolution experiment (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Bacteria were cultured on tryptic soy agar (TSA; 20\u0026deg;C, 72 hours) and tryptic soy broth (TSB; 28\u0026deg;C, 150 rpm, overnight) unless otherwise stated. A list of the bacteria used and generated for genetic manipulation in this study can be found in Supplementary Table\u0026nbsp;20, Additional file 2.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eIn preparation for transcriptomic analysis of ancestral and wrinkly MYb11, RNA was isolated from liquid cultures (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.8) and single colonies (20\u0026deg;C, 72h). For the liquid environment, a starter culture was prepared in 50 ml Falcon tubes. 10 ml TSB were inoculated with 100 \u0026micro;l of a MYb11 overnight culture and cultivated (28\u0026deg;C, 150 rpm) to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.8. 1 ml of liquid cultures were centrifuged at 6000 rpm for 5 min, and pellets or colonies were resuspended in 800 \u0026micro;l TRI-zol\u0026trade; reagent (ThermoFisher), frozen and stored (-80\u0026deg;C). RNA was isolated using the Direct-zol RNA Miniprep Kit (Zymo Research). NGS analyses were performed at the Competence Center for Genome Analysis (Kiel, Germany) using Illumina stranded total RNA library preparation and NovaSeq SP 2x50 bp sequencing.\u003c/p\u003e\n\u003ch3\u003eTranscriptome analyses\u003c/h3\u003e\n\u003cp\u003eTo determine differentially expressed genes in the host-specialized \u003cem\u003eP. lurida\u003c/em\u003e MYb11 compared to the ancestral MYb11, the RNA-Seq data were analyzed using high-performance computers at Kiel University Computer Center and R (version 4.1.1) with the following workflows and programs: Sequence quality control was assessed with FastQC (Babraham Institute) and MultiQC (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Adapter trimming was achieved with Trimmomatic (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) in paired-end mode (version 0.39, phred33, adaptersUsed\u0026thinsp;=\u0026thinsp;TruSeq3-PE-2.fa, seedMismatches\u0026thinsp;=\u0026thinsp;2, palindromeClipThreshold\u0026thinsp;=\u0026thinsp;30, simpleClipThreshold\u0026thinsp;=\u0026thinsp;10, minimumadapterlength\u0026thinsp;=\u0026thinsp;2, lengthHeadcrop\u0026thinsp;=\u0026thinsp;5, lengthMin\u0026thinsp;=\u0026thinsp;36). Read mapping and read counting were performed with EDGE-pro set for paired-end reads (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), reference genome (NZ_CP023272.1) and files for \u003cem\u003ePseudomonas lurida\u003c/em\u003e MYb11 (Pseudomonas_lurida_MYb11_6243.csv.gz) and GO annotations (\u003cem\u003ePseudomonas lurida\u003c/em\u003e MYb11 GO Term Annotations: gene_ontology_csv.csv) were obtained from Pseuomonas.com (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), mutations for the host specialists \u003cem\u003ewspE\u003c/em\u003e, \u003cem\u003ewspF\u003c/em\u003e and \u003cem\u003erph\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) were manually entered in the genome. Low read count filtering, data normalization, differential expression analysis with a negative binomial generalized linear model followed by a quasi-likelihood F-test and FDR correction for multiple testing, and Gene Set Enrichment Analysis (permutations\u0026thinsp;=\u0026thinsp;1000, minimum gene set size\u0026thinsp;=\u0026thinsp;2, and FDR correction for multiple testing) were performed using the R (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) packages edgeR version 4.2.1 (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and clusterProfiler version 4.12.6 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). KEGG Orthology was inferred using the KAAS online tool (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) (settings: GHOSTX, pep, all pseudomonas, BBH).\u003c/p\u003e\n\u003ch3\u003eMutant generation\u003c/h3\u003e\n\u003cp\u003eA two-step allele replacement procedure based on previously described protocols (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) was used to delete candidate genes in the \u003cem\u003ewspE\u003c/em\u003e host specialist background. In detail, ~\u0026thinsp;700 bp long PCR amplicons surrounding each mutation were cloned into pUISacB, allowing sucrose selection. The constructs were transformed into competent \u003cem\u003eE. coli\u003c/em\u003e cells and transferred to \u003cem\u003ewspE\u003c/em\u003e host specialists by tri parental conjugation with an \u003cem\u003eE. coli\u003c/em\u003e helper strain containing pRK2013 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Primers (Supplementary Table\u0026nbsp;21, Additional file 2) were designed using SnapGene software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.snapgene.com\u003c/span\u003e\u003cspan address=\"http://www.snapgene.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and NEBuilder v2.10.2 (New England Biolabs).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation\u003c/h2\u003e \u003cp\u003eTo determine the adherent biofilm biomass we used previously described protocols (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In detail, the bacterial cultures were adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1 with M9 buffer and diluted 1:10 with TSB. Subsequently, 180 \u0026micro;l of the dilutions were transferred to a 96-well, flat-bottomed polystyrene microtiter plate. The plates were incubated for 48 hours at 20\u0026deg;C and 125 rpm. 200 \u0026micro;l of 0.01% crystal violet was added, incubated for 30 minutes at room temperature, washed four times with 300 \u0026micro;l ddH2O, 200 \u0026micro;l of acetic acid was added and incubated for 30 minutes at room temperature. The absorbance was measured at 590 nm, 550 nm and 530 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial persistence in worms\u003c/h3\u003e\n\u003cp\u003eWe quantified the persistence in worms from the L4 stage onwards, as described before (see Early colonization, persistence and release in worms) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Bacterial lawns were prepared on NGM (125 \u0026micro;l, OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2) from ancestral and mutated MYb11 (overnight cultures: 28\u0026deg;C, 150 rpm). For persistence assays, 40 synchronized worms were raised on the respective bacteria (from L1 to L4 stage, 20\u0026deg;C). The worms were collected with M9 buffer containing 0.025% Triton-100 and 25 mM of the paralyzing antihelminthic agent tetramisole. Worms were washed in buffer using a custom-made filter tip wash system (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and then suspended in 200 \u0026micro;l of M9 and incubated for 1 hour (20\u0026deg;C), after which 100 \u0026micro;l of supernatant was collected. After homogenization with beads (1 mm zirconia), serial dilutions of worm suspension were plated and CFUs quantified. CFU/worm was calculated as the difference in CFU between worm and supernatant samples divided by the number of worms.\u003c/p\u003e\n\u003ch3\u003eCompetitive fitness in MY316 host\u003c/h3\u003e\n\u003cp\u003eCompetition experiments were performed as described for the persistence experiments and described before (see \u003cem\u003eIn vivo\u003c/em\u003e competition assays) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Co-inoculated bacteria were adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2 and mixed in equal amounts before seeding as lawns on NGM agar. A MYb11 labeled with dTomato (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) was used, which corresponds to the ancestral MYb11, as no differences were observed in short-term persistence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The competitive index was calculated as the ratio of CFU/worm of the evolved or generated mutants to CFU/worm of the ancestor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003ePrior to data analysis, the assumptions of parametric tests (normality, homogeneity of variances) were checked with Shapiro-Wilk and F-tests (Supplementary Tables\u0026nbsp;14\u0026ndash;19, Additional file 2). If these were not met, non-parametric tests were applied. FDR correction was used for multiple testing. Box plots show the median (center line), the upper/lower quartiles (box boundaries) and the 1.5-fold interquartile ranges (whiskers). All analyses and plots were performed in R/RStudio (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) using the packages ggplot2 (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), VennDiagram (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), cowplot (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), plotly (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), and dplyr (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) as well as Inkscape.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHost specialists show enriched expression of genes related to cell adhesion, flagellar function and biofilm formation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn a previous study, we showed that c-di-GMP is a key factor for the adaptation of MYb11 to its host \u003cem\u003eC. elegans\u003c/em\u003e MY316. Host adaptation was caused by mutations in the \u003cem\u003ewspE\u003c/em\u003e, \u003cem\u003ewspF\u003c/em\u003e, and \u003cem\u003erph\u003c/em\u003e, which all lead to elevated c-di-GMP levels (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, to date, the downstream targets of c-di-GMP in this adaptation remain unresolved. As a first step to identify potential downstream targets, we performed transcriptomic analyses of ancestral- versus the three host-specialized MYb11 mutants (i.e., with mutations in the genes \u003cem\u003ewspE\u003c/em\u003e, \u003cem\u003ewspF\u003c/em\u003e, and \u003cem\u003erph\u003c/em\u003e) in different culture environments, including a solid environment on agar (i.e., tryptic soy agar, TSA) and a liquid medium (tryptic soy broth, TSB). A principal component analysis (PCA) of the mapped RNA-Seq counts per million (CPM) reads of ancestral and host-specialized MYb11 in the different environments showed that 72% (PC1) of the variation could be explained by the different culture environments, while 5.7% could be explained by the genomic background of the samples (PC3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Supplementary Figs.\u0026nbsp;1\u0026ndash;3 for PC2 and PCAs for each environmental conditions and Supplementary Table\u0026nbsp;1, Additional files 1\u0026ndash;2). In this PCA, the samples from solid medium show a more compact distribution than those from liquid medium. Considering that the PCA uses expression data from all genes for the four strains, this result suggests that there is overall less variation among strains under solid conditions. For the subsequent differential gene expression, samples from different culture environments were analyzed separately to focus on the difference between the ancestral and host-specialized MYb11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb; lists of differentially expressed genes (DEG), are given in Supplementary Tables\u0026nbsp;2\u0026ndash;7, Additional file 2). The distribution of DEGs for the \u003cem\u003ewspE\u003c/em\u003e mutant compared with the ancestor are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec for the two media conditions separately. A few examples of significantly DEGs are highlighted, including those analyzed further through genetic manipulation below (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). To characterize potential downstream functions of upregulated c-di-GMP, we then focused on the overlap of DEGs among the three mutants (850 in liquid medium, 104 DEGs on solid medium, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) Supplementary Tables\u0026nbsp;8\u0026ndash;9 in Additional file 2) and subjected these gene sets to a gene set enrichment analysis (GSEA) using gene ontology (GO) terms. The results reveal eight distinct significantly enriched GO terms under liquid conditions. Of these, the GO terms cell adhesion, pilus, flagellum-dependent cell motility, flagellum basal body, and motor activity are likely important for host specialization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Supplementary Table\u0026nbsp;10 in Additional file 2). Under solid conditions, only the GO term for alginic acid biosynthesis was significantly enriched. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Supplementary Table\u0026nbsp;11, Additional file 2). Overall, the transcriptome analysis of the host-specialized \u003cem\u003eP. lurida\u003c/em\u003e MYb11 revealed that genes related to cell adhesion, such as the fimbrial genes, flagellar function and biofilm formation, such as the \u003cem\u003ealg\u003c/em\u003e and \u003cem\u003epel\u003c/em\u003e gene clusters, are potential downstream targets of c-di-GMP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003epelD\u003c/b\u003e, \u003cb\u003ebut none of the other tested candidate genes contributes to MYb11 competitive fitness and biofilm formation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn a next step, we aimed to validate the role of the differentially expressed genes as downstream targets of c-di-GMP and therefore selected genes for genetic manipulation. For this, we focused on genes with (i) significant and high differential expression, and (ii) putative functions that match those identified in the GSEA (see red labelled genes in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003efimA\u003c/em\u003e and \u003cem\u003efimD\u003c/em\u003e were selected as genes involved in pilus formation and surface adhesion (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Further, we selected \u003cem\u003eazu\u003c/em\u003e, which is linked to cell copper homeostasis and type VI secretion (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Notably, \u003cem\u003eazu\u003c/em\u003e was found to be upregulated in \u003cem\u003eP. aeruginosa\u003c/em\u003e from the lungs of patients with chronic cystic fibrosis (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The flagellar motor switch genes \u003cem\u003efliM\u003c/em\u003e and \u003cem\u003efliN\u003c/em\u003e, which are described to be involved in flagellar rotation and surface recognition (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) were chosen for the investigation of flagellar function. As genes indicative of biofilm formation and direct regulatory interaction with c-di-GMP (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), we also selected \u003cem\u003ealgD, alg44\u003c/em\u003e and \u003cem\u003epelD\u003c/em\u003e.\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\u003eCandidate genes for genetic manipulation in \u003cem\u003ewspE\u003c/em\u003e host specialist\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene product\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGO term\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene locus_tag\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLog2FC\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\u003efimA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etype 1 fimbrial protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecell adhesion / pilus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS17845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.846\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAzu\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eazurin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecopper ion binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS02765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.982\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efliM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eflagellar motor switch protein FliM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emotor activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS19420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.705\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efliN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eflagellar motor switch protein FliN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emotor activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS19415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epelD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esugar transporter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS01470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ealgD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enucleotide sugar dehydrogenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ealginic acid biosynthetic process\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS05035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efimD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efimbrial protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epilus assembly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS17855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ealg44\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ealginate biosynthesis protein Alg44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecyclic-di-GMP binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCLM75_RS05020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.639\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\u003eLegend to the table: The candidate genes have significant and high differential expression (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2FC\u0026thinsp;\u0026ge;\u0026thinsp;\u0026plusmn;\u0026thinsp;1), and/or putative functions consistent with the functions identified in the GSEA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Supplementary Tables\u0026nbsp;8\u0026ndash;11, Additional file 2). NZ_CP023272.1 was used as the reference genome for read mapping and enrichment with GO terms (Supplementary Table\u0026nbsp;12, Additional file 2); the names correspond to the KEGG Orthology (Supplementary Table\u0026nbsp;13, Additional file 2).\u003c/p\u003e \u003cp\u003eAfter selecting candidate genes for potential c-di-GMP downstream targets, we generated gene knockouts in the \u003cem\u003ewspE\u003c/em\u003e host specialist background. We focused on this specific mutant background because it showed the most robust host-specialist phenotype in various host assays (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). We examined the competitive fitness of the generated mutants in competition with the ancestral MYb11 in the natural \u003cem\u003eC. elegans\u003c/em\u003e MY316 host. Competitive fitness was performed as a short-term persistence assay and yielded data on colony-forming units (CFU) for the competing bacterial strains per worm host (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). We found that all \u003cem\u003e∆algD\u003c/em\u003e, \u003cem\u003e∆alg44\u003c/em\u003e, \u003cem\u003e∆azu\u003c/em\u003e, \u003cem\u003e∆fimA\u003c/em\u003e, \u003cem\u003e∆fliM∆fliN\u003c/em\u003e and \u003cem\u003e∆fimD\u003c/em\u003e mutants maintained significantly higher competitive fitness compared to the ancestral MYb11 and thus did not lose the high competitiveness of the \u003cem\u003ewspE\u003c/em\u003e-mutant host specialist (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea left panel, Supplementary Table\u0026nbsp;14, Additional file 2).\u003c/p\u003e \u003cp\u003eBecause the switch from a mobile to a sessile lifestyle mediated by c-di-GMP is often reflected by multiple changes in gene expression (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), we exemplarily tested whether the deletion of more than one gene was needed to reverse the host specialist phenotype. In detail, we generated \u003cem\u003e∆fimA∆fimD\u003c/em\u003e to test whether we could disrupt adhesion with multiple knockouts in the same pathway, \u003cem\u003e∆fliM∆fliN∆fimD\u003c/em\u003e to disrupt adhesion and different flagellar swimming modes, \u003cem\u003e∆pelD\u003c/em\u003e to disrupt Pel-polysaccharides as another upregulated biofilm gene cluster, and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e to disrupt Pel-polysaccharide production and adhesion. Of these mutants, \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e showed the lower ancestral competitive fitness and thus lost one of the important characteristics of the host specialist \u003cem\u003ewspE\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, right panel, Supplementary Table\u0026nbsp;14, Additional file 2).\u003c/p\u003e \u003cp\u003eIn a next step, we investigated how the mutations affect the fitness of the population within the host (total number of CFU/worm as CFU/worm mutant\u0026thinsp;+\u0026thinsp;ancestor). The mutants \u003cem\u003e∆algD\u003c/em\u003e, \u003cem\u003eΔalg44\u003c/em\u003e, \u003cem\u003e∆fliM∆fliN\u003c/em\u003e, \u003cem\u003eΔfimD\u003c/em\u003e, \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e lost the significantly increased CFU/worm number in competition compared to the ancestral MYb11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Notably, only the competitions with \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e mutants showed a significantly reduced CFU/worm number compared to \u003cem\u003ewspE\u003c/em\u003e host specialist (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Supplementary Tables\u0026nbsp;15\u0026ndash;16, Additional file 2). Similar trends, although not significant, were observed for mono-colonization of \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e mutants compared to \u003cem\u003ewspE\u003c/em\u003e host specialists (Supplementary Fig.\u0026nbsp;4, Supplementary Tables\u0026nbsp;17\u0026ndash;18, Additional files 1\u0026ndash;2).\u003c/p\u003e \u003cp\u003eIn addition, we analyzed the potential of the mutants to adhere to surfaces by forming biofilms using a crystal violet microtiter plate adherence assay. We included \u003cem\u003eE. coli\u003c/em\u003e OP50 as a negative control for biofilm formation (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). The increased adherent biofilm biomass was lost in \u003cem\u003e∆algD\u003c/em\u003e, \u003cem\u003eΔalg44\u003c/em\u003e, \u003cem\u003eΔazu\u003c/em\u003e, \u003cem\u003e∆fliM∆fliN\u003c/em\u003e, \u003cem\u003e∆pelD\u003c/em\u003e, \u003cem\u003eΔfimAΔfimD\u003c/em\u003e, \u003cem\u003e∆fliM∆fliN∆fimD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e mutants compared to ancestral \u003cem\u003eP. lurida\u003c/em\u003e MYb11. However, a significant decrease was only observed for \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e as compared to the ancestral MYb11 and the \u003cem\u003ewspE\u003c/em\u003e host specialist (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, Supplementary Table\u0026nbsp;19, Additional file 2).\u003c/p\u003e \u003cp\u003eIn summary, the \u003cem\u003e∆pelD\u003c/em\u003e and \u003cem\u003e∆pelD∆fimD\u003c/em\u003e mutants in the \u003cem\u003ewspE\u003c/em\u003e host specialist background have lost two important features of the host-specialized MYb11, namely increased host competitive fitness in the host and increased biomass of the adherent biofilm. Although other genes identified in the transcriptome analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) have already been linked to c-di-GMP-mediated shifts in Pseudomonads (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), we were only able to causally link \u003cem\u003epelD\u003c/em\u003e to the observed adaptive traits of the host-specialized \u003cem\u003eP. lurida\u003c/em\u003e MYb11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we characterized possible downstream targets of c-di-GMP-mediated adaptation of host-specialized \u003cem\u003eP. lurida\u003c/em\u003e MYb11. Our transcriptome analysis showed that the three different host specialist mutants varied from the ancestral MYb11 strain in gene expression for functions related to cell adhesion, flagellar function and biofilm formation. Our subsequent functional genetic analysis revealed that only the deletion of \u003cem\u003epelD\u003c/em\u003e was able to reverse the host specialist phenotype in the tested traits: competitive fitness in the host, total cell number in the host, and biofilm formation. Thus, our results emphasize the importance of \u003cem\u003epelD\u003c/em\u003e for the persistence and the biofilm phenotype of MYb11 host specialists, while other functions discovered in the transcriptome analyses may only play a minor role, at least at the level of a single gene.\u003c/p\u003e \u003cp\u003ePelD has been shown to post-transcriptionally regulate the production of Pel-polysaccharides in \u003cem\u003eP. aeruginosa\u003c/em\u003e in a c-di-GMP-dependent manner (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). c-di-GMP binds to the degenerate GGDEF domain of the inner membrane protein PelD, leading to a conformational change and thus enabling the biosynthesis of Pel-polysaccharides (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Functional predictions of Interpro (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) retrieved from Pseudomonas.com (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) indicate that this degenerate GGDEF domain is also part of the PelD of \u003cem\u003eP. lurida\u003c/em\u003e MYb11. Furthermore, it was previously shown that \u003cem\u003epelD\u003c/em\u003e is absolutely required for the production of Pel polysaccharides in \u003cem\u003eP. aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), suggesting that deletion of \u003cem\u003epelD\u003c/em\u003e in \u003cem\u003eP. lurida\u003c/em\u003e MYb11 also disrupts the production of Pel polysaccharides. It follows that the higher competitive fitness of host specialists, and the enhanced biofilm formation mediated by c-di-GMP are specifically and highly dependent on a functioning \u003cem\u003epel\u003c/em\u003e gene cluster and Pel-polysaccharides. Moreover, MYb11 is a Pseudomonad that harbors the major exopolysaccharide gene clusters \u003cem\u003ealg\u003c/em\u003e, \u003cem\u003ewss\u003c/em\u003e, \u003cem\u003epsl\u003c/em\u003e and \u003cem\u003epel\u003c/em\u003e (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), but our data suggest that none of the remaining gene clusters can compensate for the production of Pel-polysaccharides, indicating that Pel-polysaccharides are the major component of the adherent biofilm of MYb11.\u003c/p\u003e \u003cp\u003eThis study focused on the downstream targets of c-di-GMP that are important for host association. However, the host-specialized \u003cem\u003eP. lurida\u003c/em\u003e MYb11 evolved in a life cycle consisting of a host-associated and a free-living phase. The lack of an effect on the host association of other upregulated genes in the host-specialized MYb11 could have several reasons: Firstly, the lack of effect could simply be explained by genomic redundancy, i.e. genes in the tested signaling pathway, but also in the entire genome, could take over the lost function. This could be the case, for example, for genes involved in the production of fimbriae. In the MYb11 genome of \u003cem\u003eP. lurida\u003c/em\u003e, other genes encoding fimbrial proteins such as \u003cem\u003efimA\u003c/em\u003e and \u003cem\u003efimD\u003c/em\u003e are present (CLM75_RS01985, CLM75_RS01970 and CLM75_RS17200, Supplementary Tables\u0026nbsp;12\u0026ndash;13, Additional file 2). On the other hand, the upregulated genes might play a role for survival in a free-living environment and be less important inside the host. For example, genes related to alginate production have been shown to be important for the adaptation and survival of \u003cem\u003eP. fluorescens\u003c/em\u003e PF0-1 in dehydrated soil (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In another soil bacterium, \u003cem\u003eP. putida\u003c/em\u003e, the regulation of flagellar rotation and thus swimming mode has been shown to be important for motility in different liquid and solid environments (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In contrast, azurin is used by Pseudomonads to maintain copper-ion homeostasis in combination with TonB-dependent receptors and type VI secretion systems and therefore may play a role in nutrient acquisition in a free-living environment (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA detailed knowledge of how microbes adapt to the host association and persist in a host is crucial for full appreciation of the formation and functioning of host-microbiome interactions. Investigating the molecular basis of these adaptations in a single bacterial-host association will help us to identify conserved mechanisms as well as species-specific differences between microbes. In our work, we use \u003cem\u003eC. elegans\u003c/em\u003e MY316 and its symbiont \u003cem\u003eP. lurida\u003c/em\u003e MYb11 to study bacterial adaptation to a host. c-di-GMP and Pel-polysaccharides have been shown to be involved in host association, especially of pathogenic bacteria (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). One example is the association of \u003cem\u003eP. aeruginosa\u003c/em\u003e with cystic fibrosis lung, where Pel-polysaccharides contribute to the biofilm matrix that allows the bacteria to persist in the lung and protect it from host immune defenses and antibiotics (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). To our knowledge, Pel-polysaccharides have not yet been shown to be involved in beneficial host-microbe associations. Thus, it is conceivable that many of the mechanisms previously reported to shape the much more intensively studied interactions between host and pathogens are actually involved in any kind of symbiotic interaction, ranging from mutualistic over commensal to pathogenic associations.\u003c/p\u003e \u003cp\u003eOverall, the current study extends our previous work (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) by providing new insights into the mechanisms of c-di-GMP-driven host adaptation in symbioses beyond infection and demonstrates the specific role of \u003cem\u003epelD\u003c/em\u003e that acts downstream of c-di-GMP to mediate the competitive fitness of \u003cem\u003ePseudomonas lurida\u003c/em\u003e MYb11 within its nematode host \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e MY316.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome sequence data is available from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) under the following accession number: GSE288391. Other datasets generated and analyzed during the current study are available from our pelD_c-di-GMP_host_specialization Github repository: https://github.com/evoecogen/pelD_c-di-GMP_host_specialization.git\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Project-ID 261376515 – SFB 1182, Projects A4.3 and B4.3 (NO, HS); the DFG Clinician Scientist Program in Evolutionary Medicine „CSEM“ project 413490537 (EEG), the DFG Research Infrastructure NGS_CC project 407495230 (SF) as part of the Next Generation Sequencing Competence Network project 423957469; the International Max-Planck Research School for Evolutionary Biology (NO, AC); and the Max-Planck Society (Fellowship to HS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.C., N.O. and H.S. conceptualized the project. A.C., N.O. and E.E.G. developed the methodology. A.C., N.O., J.L. and S.F. conducted investigations. A.C., N.O. and J.L. analyzed data. A.C., N.O. and H.S. contributed to the writing of the manuscript. N.O. and H.S. supervised the project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank P. Rainey, D. Rogers, J. Summers (Max-Planck Institute for Evolutionary Biology, Ploen, Germany) for providing bacterial strains and plasmids, and advice on allelic exchange; J. Zimmermann, Daniel Schütz (Schulenburg group, University of Kiel, Germany) for bioinformatic support; J. Hofmann, H. Griem-Krey, L. Bluhm, L. Rheindorf, K. Flinder and N. Steinbach (all Schulenburg group, University of Kiel, Germany) for lab support; the Kiel BiMo/LMB for access to their core facilities; S. Koehler (Schulenburg group, University of Kiel, Germany) and H. Sondermann (Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany) for advice on the manuscript; the Schulenburg lab for project feedback.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBosch TCG, Blaser MJ, Ruby E, McFall-Ngai M. A new lexicon in the age of microbiome research. Philos Trans R Soc Lond B Biol Sci. 2024 May 6;379(1901):20230060. \u003c/li\u003e\n\u003cli\u003eKissoyan KAB, Drechsler M, Stange EL, Zimmermann J, Kaleta C, Bode HB, et al. Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr Biol. 2019 Mar;29(6):1030-1037.e5. \u003c/li\u003e\n\u003cli\u003eEngel P, Moran NA. The gut microbiota of insects - diversity in structure and function. FEMS Microbiol Rev. 2013 Sep;37(5):699\u0026ndash;735. \u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-P\u0026eacute;rez O, Cruz-Ram\u0026oacute;n V, Chinchilla-L\u0026oacute;pez P, M\u0026eacute;ndez-S\u0026aacute;nchez N. The Role of the Gut Microbiota in Bile Acid Metabolism. Ann Hepatol. 2017 Nov;16(Suppl. 1: s3-105.):s15\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eSingh A, Luallen RJ. Understanding the factors regulating host-microbiome interactions using Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 2024 May 6;379(1901):20230059. \u003c/li\u003e\n\u003cli\u003eMcFall-Ngai M. Host-Microbe Symbiosis: The Squid-Vibrio Association\u0026mdash;A Naturally Occurring, Experimental Model of Animal/Bacterial Partnerships. In: Huffnagle GB, Noverr MC, editors. GI Microbiota and Regulation of the Immune System [Internet]. New York, NY: Springer; 2008 [cited 2024 Nov 19]. p. 102\u0026ndash;12. Available from: https://doi.org/10.1007/978-0-387-09550-9_9\u003c/li\u003e\n\u003cli\u003eNyholm SV, McFall-Ngai MJ. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat Rev Microbiol. 2021 Oct;19(10):666\u0026ndash;79. \u003c/li\u003e\n\u003cli\u003eVisick KL, Stabb EV, Ruby EG. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat Rev Microbiol. 2021 Oct;19(10):654\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eKremer N, Philipp EER, Carpentier MC, Brennan CA, Kraemer L, Altura MA, et al. Initial Symbiont Contact Orchestrates Host-Organ-wide Transcriptional Changes that Prime Tissue Colonization. Cell Host Microbe. 2013 Aug 14;14(2):183\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eKissoyan KAB, Peters L, Giez C, Michels J, Pees B, Hamerich IK, et al. Exploring Effects of C. elegans Protective Natural Microbiota on Host Physiology. Front Cell Infect Microbiol. 2022 Feb 14;12:775728. \u003c/li\u003e\n\u003cli\u003eGriem-Krey H, Petersen C, Hamerich IK, Schulenburg H. The intricate triangular interaction between protective microbe, pathogen and host determines fitness of the metaorganism. Proc Biol Sci. 2023 Dec 6;290(2012):20232193. \u003c/li\u003e\n\u003cli\u003eObeng N, Bansept F, Sieber M, Traulsen A, Schulenburg H. Evolution of Microbiota\u0026ndash;Host Associations: The Microbe\u0026rsquo;s Perspective. Trends Microbiol. 2021 Sep 1;29(9):779\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eIsenberg RY, Mandel MJ. Cyclic Diguanylate in the Wild: Roles During Plant and Animal Colonization. Annu Rev Microbiol [Internet]. 2024 Sep 13 [cited 2024 Sep 24]; Available from: https://www.annualreviews.org/content/journals/10.1146/annurev-micro-041522-101729\u003c/li\u003e\n\u003cli\u003eRobinson CD, Bohannan BJ, Britton RA. Scales of persistence: transmission and the microbiome. Curr Opin Microbiol. 2019 Aug 1;50:42\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eR\u0026ouml;mling U, Galperin MY, Gomelsky M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev MMBR. 2013 Mar;77(1):1\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003eHa DG, O\u0026rsquo;Toole GA. c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.MB-0003\u0026ndash;2014. \u003c/li\u003e\n\u003cli\u003eLaventie BJ, Jenal U. Surface Sensing and Adaptation in Bacteria. Annu Rev Microbiol. 2020 Sep 8;74:735\u0026ndash;60. \u003c/li\u003e\n\u003cli\u003eKoska M, Kordes A, Erdmann J, Willger SD, Th\u0026ouml;ming JG, B\u0026auml;hre H, et al. Distinct Long- and Short-Term Adaptive Mechanisms in Pseudomonas aeruginosa. Microbiol Spectr. 2022 Dec 21;10(6):e0304322. \u003c/li\u003e\n\u003cli\u003eRuhluel D, Fisher L, Barton TE, Leighton H, Kumar S, Amores Morillo P, et al. Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments. ISME J. 2024 Jan 8;18(1):wrae065. \u003c/li\u003e\n\u003cli\u003eFranklin M, Nivens D, Weadge J, Howell P. Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl. Front Microbiol [Internet]. 2011 [cited 2023 Dec 21];2. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2011.00167\u003c/li\u003e\n\u003cli\u003eIsenberg RY, Christensen DG, Visick KL, Mandel MJ. High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization. mBio. 2022 Aug 30;13(4):e0167122. \u003c/li\u003e\n\u003cli\u003eRobinson CD, Sweeney EG, Ngo J, Ma E, Perkins A, Smith TJ, et al. Host-emitted amino acid cues regulate bacterial chemokinesis to enhance colonization. Cell Host Microbe. 2021 Aug 11;29(8):1221-1234.e8. \u003c/li\u003e\n\u003cli\u003eHuang W, Wang D, Zhang XX, Zhao M, Sun L, Zhou Y, et al. Regulatory roles of the second messenger c-di-GMP in beneficial plant-bacteria interactions. Microbiol Res. 2024 Aug;285:127748. \u003c/li\u003e\n\u003cli\u003eObeng N, Czerwinski A, Sch\u0026uuml;tz D, Michels J, Leipert J, Bansept F, et al. Bacterial c-di-GMP has a key role in establishing host\u0026ndash;microbe symbiosis. Nat Microbiol. 2023 Aug 31;1\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eDirksen P, Marsh SA, Braker I, Heitland N, Wagner S, Nakad R, et al. The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model. BMC Biol. 2016 Dec;14(1):38. \u003c/li\u003e\n\u003cli\u003eStiernagle T. Maintenance of C. elegans. WormBook [Internet]. 2006 [cited 2020 Dec 20]; Available from: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html\u003c/li\u003e\n\u003cli\u003eEwels P, Magnusson M, Lundin S, K\u0026auml;ller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eBolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014 Aug 1;30(15):2114\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eMagoc T, Wood D, Salzberg SL. EDGE-pro: Estimated Degree of Gene Expression in Prokaryotic Genomes. Evol Bioinforma Online. 2013 Mar 10;9:127\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eWinsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FSL. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 2016 Jan 4;44(D1):D646-653. \u003c/li\u003e\n\u003cli\u003eR Core Team. R: A Language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2024. Available from: https://www.R-project.org/\u003c/li\u003e\n\u003cli\u003eChen Y, Chen L, Lun ATL, Baldoni PL, Smyth GK. edgeR 4.0: powerful differential analysis of sequencing data with expanded functionality and improved support for small counts and larger datasets [Internet]. Bioinformatics; 2024 [cited 2024 Oct 15]. Available from: http://biorxiv.org/lookup/doi/10.1101/2024.01.21.576131\u003c/li\u003e\n\u003cli\u003eXu S, Hu E, Cai Y, Xie Z, Luo X, Zhan L, et al. Using clusterProfiler to characterize multiomics data. Nat Protoc. 2024 Jul 17;1\u0026ndash;29. \u003c/li\u003e\n\u003cli\u003eMoriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007 Jul;35(Web Server issue):W182-185. \u003c/li\u003e\n\u003cli\u003eBantinaki E, Kassen R, Knight CG, Robinson Z, Spiers AJ, Rainey PB. Adaptive Divergence in Experimental Populations of \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e . III. Mutational Origins of Wrinkly Spreader Diversity. Genetics. 2007 May;176(1):441\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eHmelo LR, Borlee BR, Almblad H, Love ME, Randall TE, Tseng BS, et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc. 2015 Nov;10(11):1820\u0026ndash;41. \u003c/li\u003e\n\u003cli\u003eFigurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci. 1979 Apr 1;76(4):1648\u0026ndash;52. \u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Toole GA. Microtiter Dish Biofilm Formation Assay. J Vis Exp. 2011 Jan 30;(47):2437. \u003c/li\u003e\n\u003cli\u003ePapkou A, Guzella T, Yang W, Koepper S, Pees B, Schalkowski R, et al. The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution. Proc Natl Acad Sci U S A. 2019 Jan 15;116(3):923\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003ePosit team. RStudio: Integrated Development Environment for R [Internet]. Boston, MA: Posit Software, PBC; 2024. Available from: http://www.posit.co/\u003c/li\u003e\n\u003cli\u003eWickham H. ggplot2: Elegant Graphics for Data Analysis. 2nd ed. 2016. Cham: Springer International Publishing : Imprint: Springer; 2016. 1 p. (Use R!). \u003c/li\u003e\n\u003cli\u003eHanbo Chen. VennDiagram: Generate High-Resolution Venn and Euler Plots [Internet]. 2011 [cited 2024 Oct 15]. p. 1.7.3. Available from: https://CRAN.R-project.org/package=VennDiagram\u003c/li\u003e\n\u003cli\u003eWilke CO. cowplot: Streamlined Plot Theme and Plot Annotations for \u0026ldquo;ggplot2\u0026rdquo; [Internet]. 2015 [cited 2024 Oct 15]. p. 1.1.3. Available from: https://CRAN.R-project.org/package=cowplot\u003c/li\u003e\n\u003cli\u003eLi R, Bilal U. Interactive Web-Based Data Visualization with R, Plotly, and Shiny (Carson Sievert). Biometrics. 2021 Jun 1;77(2):776\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eWickham H, Fran\u0026ccedil;ois R, Henry L, M\u0026uuml;ller K, Vaughan D, Software P, et al. dplyr: A Grammar of Data Manipulation [Internet]. 2023 [cited 2024 Oct 15]. Available from: https://cran.r-project.org/web/packages/dplyr/index.html\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;hning J, Dobbelstein AW, Sulkowski N, Eilers K, von K\u0026uuml;gelgen A, Tarafder AK, et al. Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa. PLOS Pathog. 2023 Apr 14;19(4):e1011177. \u003c/li\u003e\n\u003cli\u003eHan Y, Wang T, Chen G, Pu Q, Liu Q, Zhang Y, et al. A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition. PLoS Pathog. 2019 Dec 2;15(12):e1008198. \u003c/li\u003e\n\u003cli\u003eWood SJ, Goldufsky JW, Seu MY, Dorafshar AH, Shafikhani SH. Pseudomonas aeruginosa Cytotoxins: Mechanisms of Cytotoxicity and Impact on Inflammatory Responses. Cells. 2023 Jan;12(1):195. \u003c/li\u003e\n\u003cli\u003eHogardt M, Heesemann J. Adaptation of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e during persistence in the cystic fibrosis lung. Int J Med Microbiol. 2010 Dec 1;300(8):557\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eGheorghita AA, Wozniak DJ, Parsek MR, Howell PL. Pseudomonas aeruginosa biofilm exopolysaccharides: assembly, function, and degradation. FEMS Microbiol Rev. 2023 Nov 1;47(6):fuad060. \u003c/li\u003e\n\u003cli\u003eValentini M, Filloux A. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria. J Biol Chem. 2016 Jun 10;291(24):12547\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003eArata Y, Oshima T, Ikeda Y, Kimura H, Sako Y. OP50, a bacterial strain conventionally used as food for laboratory maintenance of C. elegans, is a biofilm formation defective mutant. MicroPublication Biol. 2020:10.17912/micropub.biology.000216. \u003c/li\u003e\n\u003cli\u003eWhitney JC, Colvin KM, Marmont LS, Robinson H, Parsek MR, Howell PL. Structure of the Cytoplasmic Region of PelD, a Degenerate Diguanylate Cyclase Receptor That Regulates Exopolysaccharide Production in Pseudomonas aeruginosa. J Biol Chem. 2012 Jul 6;287(28):23582\u0026ndash;93. \u003c/li\u003e\n\u003cli\u003ePaysan-Lafosse T, Blum M, Chuguransky S, Grego T, Pinto BL, Salazar GA, et al. InterPro in 2022. Nucleic Acids Res. 2023 Jan 6;51(D1):D418\u0026ndash;27. \u003c/li\u003e\n\u003cli\u003eWhitfield GB, Marmont LS, Ostaszewski A, Rich JD, Whitney JC, Parsek MR, et al. Pel Polysaccharide Biosynthesis Requires an Inner Membrane Complex Comprised of PelD, PelE, PelF, and PelG. J Bacteriol. 2020 Mar 26;202(8):e00684-19. \u003c/li\u003e\n\u003cli\u003eHeredia-Ponce Z, de Vicente A, Cazorla FM, Guti\u0026eacute;rrez-Barranquero JA. Beyond the Wall: Exopolysaccharides in the Biofilm Lifestyle of Pathogenic and Beneficial Plant-Associated Pseudomonas. Microorganisms. 2021 Feb 21;9(2):445. \u003c/li\u003e\n\u003cli\u003eMarshall DC, Arruda BE, Silby MW. Alginate genes are required for optimal soil colonization and persistence by Pseudomonas fluorescens Pf0-1. Access Microbiol. 2019 May 3;1(3):e000021. \u003c/li\u003e\n\u003cli\u003ePfeifer V, Beier S, Alirezaeizanjani Z, Beta C. Role of the Two Flagellar Stators in Swimming Motility of Pseudomonas putida. mBio. 13(6):e02182-22. \u003c/li\u003e\n\u003cli\u003eValentini M, Filloux A. Multiple Roles of c-di-GMP Signaling in Bacterial Pathogenesis. Annu Rev Microbiol. 2019 Sep 8;73:387\u0026ndash;406. \u003c/li\u003e\n\u003cli\u003eChung J, Eisha S, Park S, Morris AJ, Martin I. How Three Self-Secreted Biofilm Exopolysaccharides of Pseudomonas aeruginosa, Psl, Pel, and Alginate, Can Each Be Exploited for Antibiotic Adjuvant Effects in Cystic Fibrosis Lung Infection. Int J Mol Sci. 2023 May 13;24(10):8709.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pelD, Pseudomonas lurida, Caenorhabditis elegans, c-di-GMP, biofilm, symbiosis, host-microbe interaction","lastPublishedDoi":"10.21203/rs.3.rs-5767962/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5767962/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe bacterial second messenger c-di-GMP is known to influence the formation of biofilms and thereby persistence of pathogenic and beneficial bacteria in hosts. A previous evolution experiment with \u003cem\u003ePseudomonas lurida\u003c/em\u003e MYb11, occasional symbiont of the nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e, led to the emergence of host-specialized variants with elevated intracellular c-di-GMP. Thus far, the molecular underpinnings of c-di-GMP-mediated host specialization were unknown in this symbiosis. Therefore, the current study aimed at identifying candidate molecular processes by combining transcriptomic and functional genetic analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that MYb11 host specialists differentially expressed genes related to attachment, motility and biofilm production, including \u003cem\u003epelD\u003c/em\u003e from the \u003cem\u003epel\u003c/em\u003e gene cluster. \u003cem\u003epelD\u003c/em\u003e deletion resulted in reduced intra-host competitive fitness, lower bacterial numbers in \u003cem\u003eC. elegans\u003c/em\u003e and loss of biofilm biomass.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur results identify \u003cem\u003epelD\u003c/em\u003e as a previously unknown key modulator of beneficial symbiont-host associations that acts downstream of c-di-GMP.\u003c/p\u003e","manuscriptTitle":"pelD is required downstream of c-di-GMP for host specialization of Pseudomonas lurida","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 06:56:14","doi":"10.21203/rs.3.rs-5767962/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-01T21:35:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T04:32:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274657381033117411667968023803411772721","date":"2025-03-22T12:50:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243426563113598304353185910981976782112","date":"2025-03-20T06:49:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T06:33:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T02:58:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-03-19T11:38:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cde47f74-57ef-45ce-a5b6-3495a7b9e4bd","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T15:58:15+00:00","versionOfRecord":{"articleIdentity":"rs-5767962","link":"https://doi.org/10.1186/s12866-025-03945-1","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-04-16 15:56:51","publishedOnDateReadable":"April 16th, 2025"},"versionCreatedAt":"2025-03-21 06:56:14","video":"","vorDoi":"10.1186/s12866-025-03945-1","vorDoiUrl":"https://doi.org/10.1186/s12866-025-03945-1","workflowStages":[]},"version":"v1","identity":"rs-5767962","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5767962","identity":"rs-5767962","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}