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Abstract Background The aerobic fast-growing Mycobacterium smegmatis, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior. Results In a library generated by transposon mutagenesis, we searched for M. smegmatis mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the uvrA gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively. Conclusions Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.

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., (© 2023. The Author(s).)

Background: Perturbations of animal-associated microbiomes from chemical stress can affect host physiology and health. While dysbiosis induced by antibiotic treatments and disease is well known, chemical, nonantibiotic drugs have recently been shown to induce changes in microbiome composition, warranting further exploration. Loperamide is an opioid-receptor agonist widely prescribed for treating acute diarrhea in humans. Loperamide is also used as a tool to study the impact of bowel dysfunction in animal models by inducing constipation, but its effect on host-associated microbiota is poorly characterized., Results: We used conventional and gnotobiotic larval zebrafish models to show that in addition to host-specific effects, loperamide also has anti-bacterial activities that directly induce changes in microbiota diversity. This dysbiosis is due to changes in bacterial colonization, since gnotobiotic zebrafish mono-colonized with bacterial strains sensitive to loperamide are colonized up to 100-fold lower when treated with loperamide. Consistently, the bacterial diversity of gnotobiotic zebrafish colonized by a mix of 5 representative bacterial strains is affected by loperamide treatment., Conclusion: Our results demonstrate that loperamide, in addition to host effects, also induces dysbiosis in a vertebrate model, highlighting that established treatments can have underlooked secondary effects on microbiota structure and function. This study further provides insights for future studies exploring how common medications directly induce changes in host-associated microbiota. Video Abstract., (© 2023. The Author(s).)

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: a provisional patent application has been filed: “bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains” by J.-M.G, D.P.-P. and J.B.-B. The other authors declare no conflict of interest in relation to the submitted work.

Escherichia coli is a commensal or pathogenic bacterium that can survive in diverse environments. Adhesion to surfaces is essential for E. coli colonization, and thus it is important to understand the molecular mechanisms that promote this process in different niches. Autotransporter proteins are a class of cell-surface factor used by E. coli for adherence. Here we characterized the regulation and function of YeeJ, a poorly studied but widespread representative from an emerging class of autotransporter proteins, the inverse autotransporters (IAT). We showed that the yeeJ gene is present in ~40% of 96 completely sequenced E. coli genomes and that YeeJ exists as two length variants, albeit with no detectable functional differences. We demonstrated that YeeJ promotes biofilm formation in different settings through exposition at the cell-surface. We also showed that YeeJ contains a LysM domain that interacts with peptidoglycan and thus assists its localization into the outer membrane. Additionally, we identified the Polynucleotide Phosphorylase PNPase as a repressor of yeeJ transcription. Overall, our work provides new insight into YeeJ as a member of the recently defined IAT class, and contributes to our understanding of how commensal and pathogenic E. coli colonise their environments.

Airborne chemical signals emitted by bacteria influence the behavior of other bacteria and plants. We present an overview of in vitro methods for evaluating bacterial and plant responses to bacterial volatile compounds (BVCs). Three types of equipment have been used to physically separate the bacterial test strains from either other bacterial strains or plants (in our laboratory we use either Arabidopsis or tobacco plant seedlings): a Petri dish containing two compartments (BI Petri dish); two Petri dishes connected with tubing; and a microtiter-based assay. The optimized procedure for the BI Petri dish system is described in this protocol and can be widely used for elucidation of potential function in interactions between diverse microbes and those plant and chemical volatiles emitted by bacteria that are most likely to mediate bacterial or plant responses to BVCs. We also describe a procedure for metabolome-based BVC profiling via dynamic (i.e., continuous airflow) or static headspace sampling using solid-phase microextraction (SPME). Using both these procedures, bacteria-bacteria communications and bacteria-plant interactions mediated by BVCs can be rapidly investigated (within 1-4 weeks).

Bacterial metabolism has been studied primarily in liquid cultures, and exploration of other natural growth conditions may reveal new aspects of bacterial biology. Here, we investigate metabolic changes occurring when Escherichia coli grows as surface-attached biofilms, a common but still poorly characterized bacterial lifestyle. We show that E. coli adapts to hypoxic conditions prevailing within biofilms by reducing the amino acid threonine into 1-propanol, an important industrial commodity not known to be naturally produced by Enterobacteriaceae. We demonstrate that threonine degradation corresponds to a fermentation process maintaining cellular redox balance, which confers a strong fitness advantage during anaerobic and biofilm growth but not in aerobic conditions. Whereas our study identifies a fermentation pathway known in Clostridia but previously undocumented in Enterobacteriaceae, it also provides novel insight into how growth in anaerobic biofilm microenvironments can trigger adaptive metabolic pathways edging out competition with in mixed bacterial communities.

Bacteria produce and release a large diversity of small molecules including organic and inorganic volatile compounds, hereafter referred to as bacterial volatile compounds (BVCs). Whereas BVCs were often only considered as wasted metabolic by-product sometimes perceived by animal olfactory systems, it is increasingly clear that they can also mediate cross-kingdom interactions with fungi, plants and animals. Recently, in vitro studies also reported the impact of BVCs on bacterial biology through modulation of antibiotic resistance, biofilm formation and virulence. Here, we review BVCs influence on bacterial adaptation to their environment and discuss the biological relevance of recently reported inter- and intra-species bacterial interactions mediated by BVCs.

Bacterial interactions with neighboring microorganisms via production of small metabolites enable bacteria to respond and adapt to environmental changes. The study of intercellular interactions primarily focused on soluble metabolites, but bacteria also produce and release into their headspace a wide variety of volatile secondary metabolites, the ecological roles of which have generally been overlooked. However, bacterial volatile compounds are known to contribute to interkingdom interactions (plant, fungi and nematodes), and recent studies also identified their at-a-distance influence on bacterial behavior. The present review describes the biological roles of bacterial volatile compounds in inter- and intraspecies bacterial interactions, a new and yet unexplored research area, with potential clinical and industrial applications., (© FEMS 2015. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Bacteria release a wide diversity of small bioactive molecules that often correspond to secondary metabolites. Among them, volatile molecules produced under various growth conditions were shown to mediate cross-kingdom interactions with plants, nematodes, and fungi. Although the role of volatile compounds in bacterial biology is not well understood, recent reports indicated that they could play a role in airborne interactions between bacteria and influence antibiotic resistance, biofilm formation, and virulence. In this study, we investigated long-distance effects of 14 previously described Escherichia coli volatile compounds upon the bacteria E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. We show that several of these molecules constitute chemical cues influencing growth, adhesion, and motility in exposed bacteria. Moreover, we show that aerial exposure to trimethylamine (TMA), a volatile compound produced in animal intestines and tissues upon biogenic reduction of trimethylamine oxide (TMAO), modifies the antibiotic resistance profiles of all tested Gram-positive and Gram-negative bacteria. We demonstrate that the TMA mode of action is distinct from that previously described for ammonia and results from nonspecific transient alteration of antibiotic uptake due to pH increase in the environment of bacteria aerially exposed to TMA. Our study therefore presents a new way by which volatile compounds can affect community behavior and structure in physically separated bacteria. It further demonstrates that bacterial gases and volatile compounds mediate chemical interactions, triggering functional responses that play an important role in the development of bacterial communities., Importance: Bacteria release many different volatile compounds during food transformation and fermentation. Here we sought to investigate the role of several bacterial volatile molecules released by Escherichia coli during long-distance airborne interactions with other bacteria. While several tested volatiles affect bacterial motility and surface adhesion, we show that aerial exposure to trimethylamine, a molecule produced by E. coli and many other Gram-negative bacteria in animal intestines and infected tissues, also modulates antibiotic resistance in all tested bacteria. We demonstrate that exposure to trimethylamine increases the pH of the growth medium of exposed bacteria, resulting in modifications in antibiotic uptake and transient alteration of antibiotic resistance. Our study therefore presents a new mechanism by which volatile compounds can affect community behavior and structure in physically separated bacteria, and it illustrates how airborne chemical interactions between bacteria contribute to the development of bacterial communities.

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ(E) envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S4 dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.

Background: The aerobic fast-growing Mycobacterium smegmatis, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior., Results: In a library generated by transposon mutagenesis, we searched for M. smegmatis mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the uvrA gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively., Conclusions: Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.