Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes.

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group have recently been shown to lack well-characterised OM attachment systems, but instead have OmpM, which could represent an ancestral tethering system in bacteria. Here, we have determined the structure of the most abundant OmpM protein from Veillonella parvula (diderm Firmicutes) by single particle cryogenic electron microscopy. We also characterised the channel properties of the transmembrane ß-barrel of OmpM and investigated the structure and PG-binding properties of its periplasmic stalk region. Our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula, and probably other diderm Terrabacteria. This dual function of OmpM may have played a role in the loss of the OM in ancestral bacteria and the emergence of monoderm bacterial lineages.

The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope bridge for phospholipid trafficking.

E. coli and most other diderm bacteria (those with two membranes) have an inner membrane enriched in glycerophospholipids (GPLs) and an asymmetric outer membrane (OM) containing GPLs in its inner leaflet and primarily lipopolysaccharides in its outer leaflet. In E. coli, this lipid asymmetry is maintained by the Mla system which consists of six proteins: the OM lipoprotein MlaA extracts GPLs from the outer leaflet, and the periplasmic chaperone MlaC transfers them across the periplasm to the inner membrane complex MlaBDEF. However, GPL trafficking still remains poorly understood, and has only been studied in a handful of model species. Here, we investigate GPL trafficking in Veillonella parvula, a diderm Firmicute with an Mla system that lacks MlaA and MlaC, but contains an elongated MlaD. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry which can be suppressed by mutations in tamB, supporting that these two systems have opposite GPL trafficking functions across diverse bacterial lineages. Structural modelling and subcellular localisation assays suggest that V. parvula MlaD forms a transenvelope bridge, comprising a typical inner membrane-localised MCE domain and, in addition, an outer membrane ß-barrel. Phylogenomic analyses indicate that this elongated MlaD type is widely distributed across diderm bacteria and likely forms part of the ancestral functional core of the Mla system, which would be composed of MlaEFD only.

Escherichia coli SPFH Membrane Microdomain Proteins HflKC Contribute to Aminoglycoside and Oxidative Stress Tolerance.

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

Bacterial capsular polysaccharides with antibiofilm activity share common biophysical and electrokinetic properties.

Bacterial biofilms are surface-attached communities that are difficult to eradicate due to a high tolerance to antimicrobial agents. The use of non-biocidal surface-active compounds to prevent the initial adhesion and aggregation of bacterial pathogens is a promising alternative to antibiotic treatments and several antibiofilm compounds have been identified, including some capsular polysaccharides released by various bacteria. However, the lack of chemical and mechanistic understanding of the activity of these polymers limits their use to control biofilm formation. Here, we screen a collection of 31 purified capsular polysaccharides and first identify seven new compounds with non-biocidal activity against Escherichia coli and/or Staphylococcus aureus biofilms. We measure and theoretically interpret the electrophoretic mobility of a subset of 21 capsular polysaccharides under applied electric field conditions, and we show that active and inactive polysaccharide polymers display distinct electrokinetic properties and that all active macromolecules share high intrinsic viscosity features. Despite the lack of specific molecular motif associated with antibiofilm properties, the use of criteria including high density of electrostatic charges and permeability to fluid flow enables us to identify two additional capsular polysaccharides with broad-spectrum antibiofilm activity. Our study therefore provides insights into key biophysical properties discriminating active from inactive polysaccharides. The characterization of a distinct electrokinetic signature associated with antibiofilm activity opens new perspectives to identify or engineer non-biocidal surface-active macromolecules to control biofilm formation in medical and industrial settings.

Characterization of Pseudomonas aeruginosa l,d-Transpeptidases and Evaluation of Their Role in Peptidoglycan Adaptation to Biofilm Growth.

Peptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family and by l,d-transpeptidases (LDTs), which are dispensable for growth in most bacterial species and whose physiological functions remain elusive. In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (LdtPae1), the anchoring of lipoprotein OprI to the peptidoglycan (LdtPae2), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (LdtPae3). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by LdtPae1 and a greater extent of OprI anchoring to peptidoglycan, which is controlled by LdtPae2 and LdtPae3. Consistently, deletion of each of the ldt genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm. IMPORTANCE Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections.

Escherichia coli Aggregates Mediated by Native or Synthetic Adhesins Exhibit Both Core and Adhesin-Specific Transcriptional Responses.

Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or the presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific setup of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to nonaggregated bacteria. Our results show that, following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (antigen 43), the transcriptomic response of the bacteria was more exaggerated than that of aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights into the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. IMPORTANCE The formation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in various changes in bacterial physiology. However, whether adhesin-adhesin interactions, as well as the type of adhesin mediating aggregation, affect bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and nonaggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.

Intermittent antibiotic treatment of bacterial biofilms favors the rapid evolution of resistance.

Bacterial antibiotic resistance is a global health concern of increasing importance and intensive study. Although biofilms are a common source of infections in clinical settings, little is known about the development of antibiotic resistance within biofilms. Here, we use experimental evolution to compare selection of resistance mutations in planktonic and biofilm Escherichia coli populations exposed to clinically relevant cycles of lethal treatment with the aminoglycoside amikacin. Consistently, mutations in sbmA, encoding an inner membrane peptide transporter, and fusA, encoding the essential elongation factor G, are rapidly selected in biofilms, but not in planktonic cells. This is due to a combination of enhanced mutation rate, increased adhesion capacity and protective biofilm-associated tolerance. These results show that the biofilm environment favors rapid evolution of resistance and provide new insights into the dynamic evolution of antibiotic resistance in biofilms.

Pseudomonas aeruginosa Production of Hydrogen Cyanide Leads to Airborne Control of Staphylococcus aureus Growth in Biofilm and In Vivo Lung Environments.

Diverse bacterial volatile compounds alter bacterial stress responses and physiology, but their contribution to population dynamics in polymicrobial communities is not well known. In this study, we showed that airborne volatile hydrogen cyanide (HCN) produced by a wide range of Pseudomonas aeruginosa clinical strains leads to at-a-distance in vitro inhibition of the growth of a wide array of Staphylococcus aureus strains. We determined that low-oxygen environments not only enhance P. aeruginosa HCN production but also increase S. aureus sensitivity to HCN, which impacts P. aeruginosa-S. aureus competition in microaerobic in vitro mixed biofilms as well as in an in vitro cystic fibrosis lung sputum medium. Consistently, we demonstrated that production of HCN by P. aeruginosa controls S. aureus growth in a mouse model of airways coinfected by P. aeruginosa and S. aureus. Our study therefore demonstrates that P. aeruginosa HCN contributes to local and distant airborne competition against S. aureus and potentially other HCN-sensitive bacteria in contexts relevant to cystic fibrosis and other polymicrobial infectious diseases. IMPORTANCE Airborne volatile compounds produced by bacteria are often only considered attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here, we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa strains controls Staphylococcus aureus growth in low-oxygen in vitro biofilms or aggregates and in vivo lung environments. These results are of pathophysiological relevance, since lungs of cystic fibrosis patients are known to present microaerobic areas and to be commonly associated with the presence of S. aureus and P. aeruginosa in polymicrobial communities. Our study therefore provides insights into how a bacterial volatile compound can contribute to the exclusion of S. aureus and other HCN-sensitive competitors from P. aeruginosa ecological niches. It opens new perspectives for the management or monitoring of P. aeruginosa infections in lower-lung airway infections and other polymicrobial disease contexts.

An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition.

Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun's lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.

Selection for nonspecific adhesion is a driver of FimH evolution increasing Escherichia coli biofilm capacity.

Bacterial interactions with surfaces rely on the coordinated expression of a vast repertoire of surface-exposed adhesins. However, how bacteria dynamically modulate their adhesion potential to achieve successful surface colonization is not yet well understood. Here, we investigated changes in adhesion capacity of an initially poorly adherent Escherichia coli strain using experimental evolution and positive selection for mutations improving adhesion and biofilm formation on abiotic surfaces. We showed that all identified evolved populations and clones acquired mutations located almost exclusively in the lectin domain of fimH, the gene coding for the α-d-mannose-specific tip adhesin of type 1 fimbriae, a key E. coli virulence factor. While most of these fimH mutants showed reduced mannose-binding ability, they all displayed enhanced binding to abiotic surfaces, indicating a trade-off between FimH-mediated specific and nonspecific adhesion properties. Several of the identified mutations were already reported in the FimH lectin domain of pathogenic and environmental E. coli, suggesting that, beyond pathoadaptation, FimH microevolution favoring nonspecific surface adhesion could constitute a selective advantage for natural E. coli isolates. Consistently, although E. coli deleted for the fim operon still evolves an increased adhesion capacity, mutants selected in the ∆fim background are outcompeted by fimH mutants revealing clonal interference for adhesion. Our study therefore provides insights into the plasticity of E. coli adhesion potential and shows that evolution of type 1 fimbriae is a major driver of the adaptation of natural E. coli to colonization.

Lifestyle-specific S-nitrosylation of protein cysteine thiols regulates Escherichia coli biofilm formation and resistance to oxidative stress.

Communities of bacteria called biofilms are characterized by reduced diffusion, steep oxygen, and redox gradients and specific properties compared to individualized planktonic bacteria. In this study, we investigated whether signaling via nitrosylation of protein cysteine thiols (S-nitrosylation), regulating a wide range of functions in eukaryotes, could also specifically occur in biofilms and contribute to bacterial adaptation to this widespread lifestyle. We used a redox proteomic approach to compare cysteine S-nitrosylation in aerobic and anaerobic biofilm and planktonic Escherichia coli cultures and we identified proteins with biofilm-specific S-nitrosylation status. Using bacterial genetics and various phenotypic screens, we showed that impairing S-nitrosylation in proteins involved in redox homeostasis and amino acid synthesis such as OxyR, KatG, and GltD altered important biofilm properties, including motility, biofilm maturation, or resistance to oxidative stress. Our study therefore revealed that S-nitrosylation constitutes a physiological basis underlying functions critical for E. coli adaptation to the biofilm environment.

On the strong connection between nanoscale adhesion of Yad fimbriae and macroscale attachment of Yad-decorated bacteria to glycosylated, hydrophobic and hydrophilic surfaces.

Yad fimbriae are currently viewed as versatile bacterial adhesins able to significantly mediate host or plant-pathogen recognition and contribute to the persistence of Escherichia coli in both the environment and within hosts. To date, however, the underlying adhesion process of Yad fimbriae on surfaces defined by controlled coating chemistries has not been evaluated on the relevant molecular scale. In this work, the interaction forces operational between Yad fimbriae expressed by genetically modified E. coli and self-assembled monolayers (SAM) differing in terms of charge, hydrophobicity or the nature of decorating sugar units are quantified by Single Molecule Force Spectroscopy (SMFS) on the nanoscale. It is found that the adhesion of Yad fimbriae onto probes functionalized with xylose is as strong as that measured with probes decorated with anti-Yad antibodies (ca. 80 to 300 pN). In contrast, the interactions of Yad with galactose, lactose, mannose, -OH, -NH2, -COOH and -CH3 terminated SAMs are clearly non-specific. Interpretation of SMFS measurements on the basis of worm-like-chain modeling for polypeptide nanomechanics further leads to the estimates of the maximal extension of Yad fimbriae upon stretching, of their persistence length and of their polydispersity. Finally, we show for the first time a strong correlation between the adhesion properties of Yad-decorated bacteria determined from conventional macroscopic counting methods and the molecular adhesion capacity of Yad fimbriae. This demonstration advocates the effort that should be made to understand on the nanoscale level the interactions between fimbriae and their cognate ligands. The results could further help the design of potential anti-adhesive molecules or surfaces to better fight against the virulence of bacterial pathogens.

Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition.

The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST.

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.

Autotransporters Drive Biofilm Formation and Autoaggregation in the Diderm Firmicute Veillonella parvula.

The Negativicutes are a clade of the Firmicutes that have retained the ancestral diderm character and possess an outer membrane. One of the best studied Negativicutes, Veillonella parvula, is an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial communities, including the gut and the dental plaque microbiota. Whereas the adhesion and biofilm capacities of V. parvula are expected to be crucial for its maintenance and development in these environments, studies of V. parvula adhesion have been hindered by the lack of efficient genetic tools to perform functional analyses in this bacterium. Here, we took advantage of a recently described naturally transformable V. parvula isolate, SKV38, and adapted tools developed for the closely related Clostridia spp. to perform random transposon and targeted mutagenesis to identify V. parvula genes involved in biofilm formation. We show that type V secreted autotransporters, typically found in diderm bacteria, are the main determinants of V. parvula autoaggregation and biofilm formation and compete with each other for binding either to cells or to surfaces, with strong consequences for V. parvula biofilm formation capacity. The identified trimeric autotransporters have an original structure compared to classical autotransporters identified in Proteobacteria, with an additional C-terminal domain. We also show that inactivation of the gene coding for a poorly characterized metal-dependent phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for further molecular characterization of V. parvula interactions with other bacteria and the host within complex microbiota environments.IMPORTANCEVeillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to adhere to surfaces or other bacteria and form biofilms is critical for it to inhabit complex human microbial communities such as the gut and oral microbiota. Although the adhesive capacity of V. parvula has been previously described, very little is known about the underlying molecular mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to identify surface-exposed adhesins called autotransporters as the main molecular determinants of adhesion in this bacterium. This work therefore provides new insights on an important aspect of the V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of biofilm formation to the biology of this major commensal of the oral-digestive tract.

Capsular Polysaccharide Cross-Regulation Modulates Bacteroides thetaiotaomicron Biofilm Formation.

Bacteroides thetaiotaomicron is one of the most abundant gut symbiont species, whose contribution to host health through its ability to degrade dietary polysaccharides and mature the immune system is under intense scrutiny. In contrast, adhesion and biofilm formation, which are potentially involved in gut colonization and microbiota structure and stability, have hardly been investigated in this intestinal bacterium. To uncover B. thetaiotaomicron biofilm-related functions, we performed a transposon mutagenesis in the poorly biofilm-forming reference strain VPI-5482 and showed that capsule 4, one of the eight B. thetaiotaomicron capsules, hinders biofilm formation. We then showed that the production of capsules 1, 2, 3, 5, and 6 also inhibits biofilm formation and that decreased capsulation of the population correlated with increased biofilm formation, suggesting that capsules could be masking adhesive surface structures. In contrast, we showed that capsule 8 displayed intrinsic adhesive properties. Finally, we demonstrated that BT2934, the wzx homolog of the B. thetaiotaomicron glycosylation locus, competes with capsule production and impacts its adhesion capacity. This study therefore establishes B. thetaiotaomicron capsule regulation as a major determinant of B. thetaiotaomicron biofilm formation, providing new insights into how modulation of different B. thetaiotaomicron surface structures affects in vitro biofilm formation.IMPORTANCE The human gut harbors a complex bacterial community that plays important roles in host health and disease, including nutrient acquisition, maturation of the immune system, and resistance to infections. The capacity to adhere to surfaces and form communities called biofilms is believed to be important for niche colonization and maintenance of gut bacteria. However, little is known about the adhesion capacity of most gut bacteria. In this study, we investigated biofilm formation in Bacteroides thetaiotaomicron, one of the most abundant bacteria of the normal mammalian intestine. We identified that B. thetaiotaomicron capsules, a group of eight surface-exposed polysaccharidic layers mediating important interactions with the gut environment, are also major determinants of biofilm formation that mask or unmask adhesion factors. Studying how B. thetaiotaomicron regulates its adhesion properties will allow us to better understand the physiology and specific properties of this important gut symbiont within anaerobic biofilms.

One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide.

How, when and why the transition between cell envelopes with one membrane (Gram-positives or monoderms) and two (Gram-negative or diderms) occurred in Bacteria is a key unanswered question in evolutionary biology. Different hypotheses have been put forward, suggesting that either the monoderm or the diderm phenotype is ancestral. The existence of diderm members in the classically monoderm Firmicutes challenges the Gram-positive/Gram-negative divide and provides a great opportunity to tackle the issue. In this review, we present current knowledge on the diversity of bacterial cell envelopes, including these atypical Firmicutes. We discuss how phylogenomic analysis supports the hypothesis that the diderm cell envelope architecture is an ancestral character in the Firmicutes, and that the monoderm phenotype in this phylum arose multiple times independently by loss of the outer membrane. Given the overwhelming distribution of diderm phenotypes with respect to monoderm ones, this scenario likely extends to the ancestor of all bacteria. Finally, we discuss the recent development of genetic tools for Veillonella parvula, a diderm Firmicute member of the human microbiome, which indicates it as an emerging new experimental model to investigate fundamental aspects of the diderm/monoderm transition.