Achieving completeness of multipartite bacterial genomes has been a difficult task, especially in rhizobia. In this study, we performed a deep bioinformatic analysis of the newly re-sequenced genome of Rhizobium favelukesii LPU83T. This strain was isolated from acid soils in Argentina and is capable of nodulating several leguminous plants, although it is unable to fix nitrogen efficiently in any of them. Oxford Nanopore sequencing allowed us to completely assemble the symbiotic plasmid of the strain, pRfaLPU83b, and we discovered that it harbors three intact prophages and a high density of insertion sequences (ISs). These characteristics show why it is often so difficult to complete the symbiotic plasmids of rhizobial strains and the importance of having long-read sequencing methods. Upon detailed analysis of this replicon, we identified a complete conjugation system with gene structure consistent with quorum sensing-associated systems that may have contributed to the genetic mosaic structure of the strain. Furthermore, we identified in the symbiotic plasmid of R. favelukesii LPU83T a large proportion of the symbiotic genes previously identified as essential for Biological Nitrogen Fixation (BNF) in symbiosis with alfalfa, with a high percentage of identity with respect to those of Sinorhizobium meliloti 2011. Among the determinants related to BNF, we found genes encoding the HrrP and SapA peptidases in the LPU83 genome, previously described and related to the degradation of nodule-specific cysteine-rich peptides. These peptides are essential for bacteroid differentiation and, therefore, efficient BNF. Our results show that despite having these genes, they are not directly responsible for the inefficient BNF phenotype of LPU83.

Plasmid conjugative transfer (CT) is a major mechanism of horizontal gene transfer in bacteria, facilitating genome evolution and dissemination of adaptive traits. Due to the energetic cost of CT, its regulation becomes an important process to ensure energetic balance within cells. In Rhizobium favelukesii, the plasmid pLPU83a belongs to group I-C of rhizobial plasmids, which require the transcriptional regulator TraR for CT. In well-characterized systems, TraR typically activates conjugative genes in response to quorum-sensing (QS) signals such as acyl-homoserine lactones. However, pLPU83a does not respond to these signals, raising questions about how TraR is regulated in this system. This study addresses the function of RcgA and RcgR, two proteins encoded upstream of traR on pLPU83a, whose function has previously been associated with CT modulation. Through proteomic, transcriptomic, and microscopy approaches, we show that RcgR acts as a repressor of CT, inhibiting traR expression and, therefore, the transcription of genes involved in CT, thereby reducing plasmid transfer rate. In contrast, RcgA is essential for CT but does not affect the expression of CT genes; it is localized at the membrane and may play a structural role in the mating pair formation system. Functional assays revealed that the repression facilitated by RcgR is independent of the anti-activator TraM and that TraR is essential for transfer even in the absence of RcgR. These findings locate RcgA and RcgR as key elements of a new circuit that modulates rhizobial plasmid conjugation and propose a novel mechanism of TraR control in systems uncoupled from QS signaling.IMPORTANCEPlasmid transfer is a central mechanism of gene exchange in bacteria, enabling the spread of traits with ecological and evolutionary relevance. Rhizobium favelukesii is a soil bacterium that carries multiple plasmids, including pLPU83a, which serves as a model to study conjugative transfer. This plasmid requires the transcriptional regulator TraR for transfer but-unlike classical systems-lacks the cognate gene that encodes the AHL synthase typically involved in quorum-sensing regulation. In previous work, two novel proteins encoded on pLPU83a, RcgA and RcgR, were identified as key elements in this regulatory system. Here, we further characterized their roles: RcgR represses the transcription of traR and, consequently, that of all conjugative genes, while RcgA is essential for transfer and localizes to the membrane, suggesting a structural function. These results provide mechanistic insight into how plasmid transfer is regulated in systems uncoupled from quorum sensing, highlighting alternative layers of control in bacterial conjugation.

The γ-proteobacterium Xanthomonas campestris pv. campestris B100 is the causal agent of black rot disease in a wide range of economically important crops. In addition, X. campestris pv. campestris is commercially relevant due to the synthesis of the exopolysaccharide xanthan. In this work, we first introduce a novel transcriptional regulator, termed Crt1, and present the effect that the deletion of the crt1 gene exerts on growth, xanthan production, virulence, and transcriptome and proteome profiles. Differential transcriptome analysis of the deletion mutant X. campestris pv. campestris B100 Δcrt1 compared to the wild type revealed the up-regulation of the pilP, pilM and pilE genes, which are relevant for the type 4 pilus assembly. Furthermore, increased xanthan production and upregulated transcription of genes within the gum cluster, which are critical for xanthan biosynthesis, were observed. Profiling of the cytosolic proteome identified increased expression of the glucosyltransferase GtrB, which is involved in LPS biosynthesis, and of a type III effector with phytase activity. Moreover, the presence of the fructose import and utilization proteins FruAB and FruK was reduced in the deletion mutant. The plant-pathogenicity assay demonstrated that the severity of the infection of the host plant tissue was higher in the case of the deletion mutant than in the wildtype strain. The regulator Crt1 modulates multiple virulence factors as well as LPS and xanthan production. In conclusion, Crt1 influences a diverse network of genes that contribute to the pathogenic lifestyle of X. campestris pv. campestris.

The metagenome analysis of complex environments with thousands of datasets, such as those in the Sequence Read Archive, requires substantial computational resources for it to be completed within a reasonable time frame. Efficient use of infrastructure is essential, and analyses must be fully reproducible with publicly available workflows to ensure transparency. Here, we introduce the Metagenomics-Toolkit, a scalable, data-agnostic workflow that automates the analysis of short and long metagenomic reads from Illumina and Oxford Nanopore Technology devices, respectively. The Metagenomics-Toolkit provides standard features such as quality control, assembly, binning, and annotation, along with unique capabilities including plasmid identification, recovery of unassembled microbial community members, and discovery of microbial interdependencies through dereplication, co-occurrence, and genome-scale metabolic modeling. Additionally, the Metagenomics-Toolkit includes a machine learning-optimized assembly step that adjusts peak RAM usage to match actual requirements, reducing the need for high-memory hardware. It can be executed on user workstations and includes optimizations for efficient cloud-based cluster execution. We compare the Metagenomics-Toolkit with five widely used metagenomics workflows and demonstrate its capabilities on 757 sewage metagenome datasets to investigate a possible sewage core microbiome. The Metagenomics-Toolkit is open source and available at https://github.com/metagenomics/metagenomics-tk.

Acarbose, a pseudo-tetrasaccharide produced by Actinoplanes sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in Actinoplanes sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into Actinoplanes sp. These vectors were validated by silencing the known acarbose biosynthesis genes acbB and acbV, as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein–DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production.

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σHSc. Therefore, the protein encoded by ACSP50_0507 was named σHAs. Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 (sigHAs) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σHAs frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σHAs was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σHAs to the upstream sequence of a strongly upregulated gene. Autoregulation of σHAs was observed, and binding to its own gene promoter region was also confirmed.

The pseudotetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a relevant secondary metabolite used in diabetes type II medication. Although maltose plays a crucial role in acarbose biosynthesis, the understanding of the maltose/maltodextrin metabolism and its involvement in acarbose production is at an early stage. Here, we reconstructed the predicted maltose–maltodextrin pathway that involves four enzymes AmlE, MalZ, MalP, and MalQ. An investigation of enzyme activities was conducted through in vitro assays, leading to an expansion of previously postulated substrate spectra. The maltose-induced α-glucosidase AmlE is noteworthy for its high hydrolysis rate of linear α-1,4-glucans, and its capability to hydrolyze various glycosidic bonds. The predicted maltodextrin glucosidase MalZ showed slow hydrolysis activity on linear α-glucans, but it was resistant to acarbose and capable of releasing glucose from acarbose. AmlE compensates for the low activity of MalZ to ensure glucose supply. We determined the enzyme activity of MalP and its dual function as maltodextrin and glycogen phosphorylase. The 4-α-glucanotransferase MalQ plays a central role in the maltose/maltodextrin metabolism, alongside MalP. This study confirmed the simultaneous degradation and synthesis of long-chain α-glucans. The product distribution showed that with an increasing number of glycosidic bonds, less glucose is formed. We found that MalQ, like its sequence homolog AcbQ from the acarbose biosynthetic gene cluster, is involved in the formation of elongated acarviosyl metabolites. However, MalQ does not participate in the elongation of acarbose 7-phosphate, which is likely the more readily available acceptor molecule in vivo. Accordingly, MalQ is not involved in the formation of acarviosyl impurities in Actinoplanes sp. SE50/110.

Xanthan, a bacterial polysaccharide, is widespread in industrial applications, particularly as a food additive. However, little is known about the process of xanthan synthesis on the proteome level, even though Xanthomonas campestris is frequently used for xanthan fermentation. A label-free LC-MS/MS method was employed to study the protein changes during xanthan fermentation in minimal medium. According to the reference database, 2416 proteins were identified, representing 54.75 % of the proteome. The study examined changes in protein abundances concerning the growth phase and xanthan productivity. Throughout the experiment, changes in nitrate concentration appeared to affect the abundance of most proteins involved in nitrogen metabolism, except Gdh and GlnA. Proteins involved in sugar nucleotide metabolism stay unchanged across all growth phases. Apart from GumD, GumB, and GumC, the gum proteins showed no significant changes throughout the experiment. GumD, the first enzyme in the assembly of the xanthan-repeating unit, peaked during the early stationary phase but decreased during the late stationary phase. GumB and GumC, which are involved in exporting xanthan, increased significantly during the stationary phase. This study suggests that a potential bottleneck for xanthan productivity does not reside in the abundance of proteins directly involved in the synthesis pathways.

The current focus on renewable energy in global policy highlights the importance of methane production from biomass through anaerobic digestion (AD). To improve biomass digestion while ensuring overall process stability, microbiome-based management strategies become more important. In this study, metagenomes and metaproteomes were used for metagenomically assembled genome (MAG)-centric analyses to investigate a full-scale biogas plant consisting of three differentially operated digesters. Microbial communities were analyzed regarding their taxonomic composition, functional potential, as well as functions expressed on the proteome level. Different abundances of genes and enzymes related to the biogas process could be mostly attributed to different process parameters. Individual MAGs exhibiting different abundances in the digesters were studied in detail, and their roles in the hydrolysis, acidogenesis and acetogenesis steps of anaerobic digestion could be assigned. Methanoculleus thermohydrogenotrophicum was an active hydrogenotrophic methanogen in all three digesters, whereas Methanothermobacter wolfeii was more prevalent at higher process temperatures. Further analysis focused on MAGs, which were abundant in all digesters, indicating their potential to ensure biogas process stability. The most prevalent MAG belonged to the class Limnochordia; this MAG was ubiquitous in all three digesters and exhibited activity in numerous pathways related to different steps of AD.

Microbes – bacteria, archaea, unicellular eukaryotes, and viruses – play an important role in human and environmental health. Growing awareness of this fact has led to a huge increase in microbiological research and applications in a variety of fields. Driven by technological advances that allow high-throughput molecular characterization of microbial species and communities, microbiological research now offers unparalleled opportunities to address current and emerging needs. As well as helping to address global health threats such as antimicrobial resistance and viral pandemics, it also has a key role to play in areas such as agriculture, waste management, water treatment, ecosystems remediation, and the diagnosis, treatment and prevention of various diseases. Reflecting this broad potential, billions of euros have been invested in microbiota research programs worldwide. Though run independently, many of these projects are closely related. However, Germany currently has no infrastructure to connect such projects or even compare their results. Thus, the potential synergy of data and expertise is being squandered. The goal of the NFDI4Microbiota consortium is to serve and connect this broad and heterogeneous research community by elevating the availability and quality of research results through dedicated training, and by facilitating the generation, management, interpretation, sharing, and reuse of microbial data. In doing so, we will also foster interdisciplinary interactions between researchers. NFDI4Microbiota will achieve this by creating a German microbial research network through training and community-building activities, and by creating a cloud-based system that will make the storage, integration and analysis of microbial data, especially omics data, consistent, reproducible, and accessible across all areas of life sciences. In addition to increasing the quality of microbial research in Germany, our training program will support widespread and proper usage of these services. Through this dual emphasis on education and services, NFDI4Microbiota will ensure that microbial research in Germany is synergistic and efficient, and thus excellent. By creating a central resource for German microbial research, NDFDI4Microbiota will establish a connecting hub for all NFDI consortia that work with microbiological data, including GHGA, NFDI4Biodiversity, NFDI4Agri and several others. NFDI4Microbiota will provide non-microbial specialists from these consortia with direct and easy access to the necessary expertise and infrastructure in microbial research in order to facilitate their daily work and enhance their research. The links forged through NFDI4Microbiota will not only increase the synergy between NFDI consortia, but also elevate the overall quality and relevance of microbial research in Germany.

Abstract Background For a sustainable production of food, research on agricultural soil microbial communities is inevitable. Due to its immense complexity, soil is still some kind of black box. Soil study designs for identifying microbiome members of relevance have various scopes and focus on particular environmental factors. To identify common features of soil microbiomes, data from multiple studies should be compiled and processed. Taxonomic compositions and functional capabilities of microbial communities associated with soils and plants have been identified and characterized in the past few decades. From a fertile Loess–Chernozem-type soil located in Germany, metagenomically assembled genomes (MAGs) classified as members of the phylum Thaumarchaeota/Thermoproteota were obtained. These possibly represent keystone agricultural soil community members encoding functions of relevance for soil fertility and plant health. Their importance for the analyzed microbiomes is corroborated by the fact that they were predicted to contribute to the cycling of nitrogen, feature the genetic potential to fix carbon dioxide and possess genes with predicted functions in plant-growth-promotion (PGP). To expand the knowledge on soil community members belonging to the phylum Thaumarchaeota, we conducted a meta-analysis integrating primary studies on European agricultural soil microbiomes. Results Taxonomic classification of the selected soil metagenomes revealed the shared agricultural soil core microbiome of European soils from 19 locations. Metadata reporting was heterogeneous between the different studies. According to the available metadata, we separated the data into 68 treatments. The phylum Thaumarchaeota is part of the core microbiome and represents a major constituent of the archaeal subcommunities in all European agricultural soils. At a higher taxonomic resolution, 2074 genera constituted the core microbiome. We observed that viral genera strongly contribute to variation in taxonomic profiles. By binning of metagenomically assembled contigs, Thaumarchaeota MAGs could be recovered from several European soil metagenomes. Notably, many of them were classified as members of the family Nitrososphaeraceae, highlighting the importance of this family for agricultural soils. The specific Loess-Chernozem Thaumarchaeota MAGs were most abundant in their original soil, but also seem to be of importance in other agricultural soil microbial communities. Metabolic reconstruction of Switzerland_1_MAG_2 revealed its genetic potential i.a. regarding carbon dioxide (CO $$_2$$ 2 ) fixation, ammonia oxidation, exopolysaccharide production and a beneficial effect on plant growth. Similar genetic features were also present in other reconstructed MAGs. Three Nitrososphaeraceae MAGs are all most likely members of a so far unknown genus. Conclusions On a broad view, European agricultural soil microbiomes are similarly structured. Differences in community structure were observable, although analysis was complicated by heterogeneity in metadata recording. Our study highlights the need for standardized metadata reporting and the benefits of networking open data. Future soil sequencing studies should also consider high sequencing depths in order to enable reconstruction of genome bins. Intriguingly, the family Nitrososphaeraceae commonly seems to be of importance in agricultural microbiomes.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

Xanthomonas campestris strains are used world-wide for the production of the industrially important exopolysaccharide xanthan. The high industrial relevance of xanthan can be explained by its extraordinary qualities as rheological control agent in aqueous systems and by its stabilizing properties in suspensions and emulsions. The phytopathogen Xanthomonas campestris is a motile bacterium with one polar flagellum. The flagellum is a cost intensive structure, in terms of energy and building block consumption. Based on the assumption that inhibition of the flagellar biosynthesis and related proton driven motility might be beneficial for the xanthan production in Xcc, two genes (fliC and fliM) were mutated to inhibit the motility. Both mutants Xcc JBL007 fliC- and Xcc JBL007 fliM- showed an increased xanthan production. Remarkably, the produced xanthan from both mutants showed enhanced rheological properties. While the chemical composition of the produced xanthan of the initial and both mutant strains did not change, notable differences in persistence length could be measured via atomic force microscopy. Results presented in this study demonstrate the possibility to further improve the xanthan production by Xcc through rational strain design.

Research on biogas-producing microbial communities aims at elucidation of correlations and dependencies between the anaerobic digestion (AD) process and the corresponding microbiome composition in order to optimize the performance of the process and the biogas output. Previously, Lachnospiraceae species were frequently detected in mesophilic to moderately thermophilic biogas reactors. To analyze adaptive genome features of a representative Lachnospiraceae strain, Anaeropeptidivorans aminofermentans M3/9T was isolated from a mesophilic laboratory-scale biogas plant and its genome was sequenced and analyzed in detail. Strain M3/9T possesses a number of genes encoding enzymes for degradation of proteins, oligo- and dipeptides. Moreover, genes encoding enzymes participating in fermentation of amino acids released from peptide hydrolysis were also identified. Based on further findings obtained from metabolic pathway reconstruction, M3/9T was predicted to participate in acidogenesis within the AD process. To understand the genomic diversity between the biogas isolate M3/9T and closely related Anaerotignum type strains, genome sequence comparisons were performed. M3/9T harbors 1,693 strain-specific genes among others encoding different peptidases, a phosphotransferase system (PTS) for sugar uptake, but also proteins involved in extracellular solute binding and import, sporulation and flagellar biosynthesis. In order to determine the occurrence of M3/9T in other environments, large-scale fragment recruitments with the M3/9T genome as a template and publicly available metagenomes representing different environments was performed. The strain was detected in the intestine of mammals, being most abundant in goat feces, occasionally used as a substrate for biogas production.

This article describes some use case studies and self-assessments of FAIR status of de.NBI services to illustrate the challenges and requirements for the definition of the needs of adhering to the FAIR (findable, accessible, interoperable and reusable) data principles in a large distributed bioinformatics infrastructure. We address the challenge of heterogeneity of wet lab technologies, data, metadata, software, computational workflows and the levels of implementation and monitoring of FAIR principles within the different bioinformatics sub-disciplines joint in de.NBI. On the one hand, this broad service landscape and the excellent network of experts are a strong basis for the development of useful research data management plans. On the other hand, the large number of tools and techniques maintained by distributed teams renders FAIR compliance challenging.

Der Aufbau einer Bioinformatik-Infrastruktur in Deutschland wird vom BMBF seit dem Jahr 2015 betrieben. Dazu wurde die Fordermasnahme „Deutsches Netzwerk fur Bioinformatik-Infrastruktur (de.NBI)” (www.denbi.de) ins Leben gerufen mit der Aufgabe, Forschenden in den Lebenswissenschaften die Analyse groser Datenmengen zu ermoglichen. Nach Einrichtung der Infrastrukturbereiche Serviceangebote, Trainingskurse und Cloud-Computing steht nun eine Verstetigung dieser Bioinformatik-Infrastruktur durch Integration in die Helmholtz-Gemeinschaft an. Die Bundesregierung hat hierzu in ihrer jungsten Finanzplanung dem Forschungszentrum Julich Finanzmittel zur Verfugung gestellt, um die etablierte Bioinformatik-Infrastruktur langfristig zu betreiben.

Sinorhizobium meliloti contains the negatively charged phosphatidylglycerol and cardiolipin as well as the zwitterionic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) as major membrane phospholipids. In previous studies we had isolated S. meliloti mutants that lack PE or PC. Although mutants deficient in PE are able to form nitrogen-fixing nodules on alfalfa host plants, mutants lacking PC cannot sustain development of any nodules on host roots. Transcript profiles of mutants unable to form PE or PC are distinct; they differ from each other and they are different from the wild type profile. For example, a PC-deficient mutant of S. meliloti shows an increase of transcripts that encode enzymes required for succinoglycan biosynthesis and a decrease of transcripts required for flagellum formation. Indeed, a PC-deficient mutant is unable to swim and overproduces succinoglycan. Some suppressor mutants, that regain swimming and form normal levels of succinoglycan, are altered in the ExoS sensor. Our findings suggest that the lack of PC in the sinorhizobial membrane activates the ExoS/ChvI two-component regulatory system. ExoS/ChvI constitute a molecular switch in S. meliloti for changing from a free-living to a symbiotic life style. The periplasmic repressor protein ExoR controls ExoS/ChvI function and it is thought that proteolytic ExoR degradation would relieve repression of ExoS/ChvI thereby switching on this system. However, as ExoR levels are similar in wild type, PC-deficient mutant and suppressor mutants, we propose that lack of PC in the bacterial membrane provokes directly a conformational change of the ExoS sensor and thereby activation of the ExoS/ChvI two-component system.