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Most bacteria are protected from environmental offenses by a cell wall consisting of strong yet elastic peptidoglycan. The cell wall is essential for preserving bacterial morphology and viability, and thus the enzymes involved in the production and turnover of peptidoglycan have become preferred targets for many of our most successful antibiotics. In the past decades, Vibrio cholerae, the gram-negative pathogen causing the diarrheal disease cholera, has become a major model for understanding cell wall genetics, biochemistry, and physiology. More than 100 articles have shed light on novel cell wall genetic determinants, regulatory links, and adaptive mechanisms. Here we provide the first comprehensive review of V. cholerae’s cell wall biology and genetics. Special emphasis is placed on the similarities and differences with Escherichia coli, the paradigm for understanding cell wall metabolism and chemical structure in gram-negative bacteria.

Peptidoglycan (PG) has remained for decades in the spotlight of the never-ending battle against pathogenic bacteria as this essential bacterial structure is one of the most successful targets for antibiotics. Most of our current understanding about the composition, architecture, and dynamics of the PG relies on techniques which have experienced great technological and methodological improvements in the past years. Here we summarize recent advances in these methods with the intention to furnish a valuable resource for both PG experts and newcomers.

Peripheral nerve section with subsequent disconnection of motor neuron (MN) cell bodies from their skeletal muscle targets leads to a rapid reactive response involving the recruitment and activation of microglia. In addition, the loss of afferent synapses on MNs occurs in concomitance with microglial reaction by a process described as synaptic stripping. However, the way in which postaxotomy‐activated microglia adjacent to MNs are involved in synaptic removal is less defined. Here, we used confocal and electron microscopy to examine interactions between recruited microglial cells and presynaptic terminals in axotomized MNs between 1 and 15 days after sciatic nerve transection in mice. We did not observe any bulk engulfment of synaptic boutons by microglia. Instead, microglial cells internalized small membranous‐vesicular fragments which originated from the acute disruption of synaptic terminals involving the activation of the necroptotic pathway. The presence of abundant extracellular vesicles in the perineuronal space after axotomy, together with the increased expression of phospho‐mixed lineage kinase domain‐like protein and, later, of extracellular vesicle markers, such as CD9, CD63, and flotillin, indicate that the vesicles mainly originated in synapses and were transferred to microglia. The upregulation of Rab7 and Rab10 in microglia interacting with injured MNs, indicated the activation of endocytosis. As activated microglia and synaptic boutons displayed positive C1q immunoreactivity, a complement‐mediated opsonization may also contribute to microglial‐mediated synaptic disruption. In addition to the relevance of our data in the context of neuroinflammation and MN disease, they should also be taken into account for understanding functional recovery after peripheral nerve injury., Main Points Early after axotomy, microglia recruited near injured motor neurons, emit processes that tend to contact their afferent synaptic terminals.Extracellular vesicles resulting from necroptotic synaptic disruption are removed by microglia.

During systemic infection of susceptible hosts, Salmonella enterica colonizes the gall bladder, which contains lethal concentrations of bile salts. Recovery of Salmonella cells from the gall bladder of infected mice yields two types of isolates: (i) bile-resistant mutants; (ii) isolates that survive lethal selection without mutation. Bile-resistant mutants are recovered at frequencies high enough to suggest that increased mutation rates may occur in the gall bladder, thus providing a tentative example of stress-induced mutation in a natural environment. However, most bile-resistant mutants characterized in this study show defects in traits that are relevant for Salmonella colonization of the animal host. Mutation may thus permit short-term adaptation to the gall bladder at the expense of losing fitness for transmission to new hosts. In contrast, non mutational adaptation may have evolved as a fitness-preserving strategy. Failure of RpoS− mutants to colonize the gall bladder supports the involvement of the general stress response in non mutational adaptation.

14 páginas.- 6 figuras.- 2 tablas.- referencias.- The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.647305/full#supplementary-material, Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA− and ZapB− mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile, This study was supported by grant BIO2016-75235-P from the Spanish Ministerio de Economía, Industria y Competitividad - Agencia Estatal de Investigación and the European Regional Fund. VU was supported by a postdoctoral contract associated to grant CVI-5879 from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain. MS-R was supported by the VI Plan Propio de Investigación y Transferencia de la Universidad de Sevilla.

A general survival strategy of many life forms faced with harmful growth conditions is to enter into a non-proliferating state until conditions suitable for growth are restored. In bacteria, this survival strategy is associated with antimicrobial tolerance, chronic infections and environmental dispersion. In particular, the agent of the deadly human disease cholera,Vibrio cholerae, undergoes a morphological transition from a rod-shaped proliferative form to a spherical non-proliferating form after exposure to cold or cell wall targeting antibiotics. Growth is resumed when the adverse conditions have ceased.Here, we show that a component of the hemicellulose and pectin of terrestrial plants, L-arabinose, triggers the formation of non-proliferatingV. choleraespherical cells, which are able to return to growth when L-arabinose is removed from the growth medium. We found that the cell wall of L-arabinose treatedV. choleraecells has a peptidoglycan composition similar to the cell wall of spheroplasts and that they revert to a wild-type morphology through the formation of branched cells like L-forms. Unlike L-forms, however, they are osmo-resistant. Through a random Tn genetic screen for mutants insensitive to L-arabinose, we identified genes involved in the uptake and catabolism of galactose and in glycolysis. We hypothesize that L-arabinose is enzymatically processed by the galactose catabolism and glycolysis pathways until it is transformed in a product that cannot be further recognized byV. choleraeenzymes. Accumulation of this enzymatic by-product triggers the formation of viable non-dividing cell wall deficient spherical cells.

International audience; Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most prescribed xenobiotics for ICU patients on fitness, pathogenicity and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out and we observed that caspofungin, an antifungal agent belonging to the echinocandins family, had an important effect on E. faecium growth in vitro. We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (¼ × MIC, 8 mg/L), caspofungin had an impact on cell wall organization especially in abundance of some muropeptide precursors. By RNA-seq, it was also shown that around 20% of the transcriptome were altered in the presence of caspofungin with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., beta-(1,3)-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the pyruvate metabolism seemed to have a drastic impact on bacterial cell viability while decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of non-antibiotic xenobiotics against bacterial pathogens.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both. Agence Nationale de la Recherche ANR-15-CE11-0001-02 Centre National de la Recherche Scientifique PICS07279 French State Program "Investissements d'Avenir" ANR-11-LABX-0011

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota., The authors present a public collection of 117 bacterial isolates from the pig gut, including the description of 38 novel taxa. Interesting functions discovered in these organisms include a new fucosyltransferease and sactipeptide-like molecules encoded by biosynthetic gene clusters.

Summary: The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for the maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized and recycled. Here, we show that Vibrio cholerae recycles PG fragments modified with non-canonical d-amino acids (NCDAA), which lead to the accumulation of cytosolic PG tetrapeptides. We demonstrate that the accumulation of recycled tetrapeptides has two regulatory consequences for the cell wall: reduction of d,d-cross-linkage and reduction of PG synthesis. We further demonstrate that l,d-carboxypeptidases from five different species show a preferential activity for substrates containing canonical (d-alanine) versus non-canonical (d-methionine) d-amino acids, suggesting that the accumulation of intracellular tetrapeptides in NCDAA-rich environments is widespread. Collectively, this work reveals a regulatory role of NCDAA linking PG recycling and synthesis to promote optimal cell wall assembly and composition in the stationary phase. : A critical step in peptidoglycan (PG) recycling is the transformation of PG tetrapeptides into tripeptides. Hernández et al. demonstrate that Vibrio cholerae accumulates tetrapeptide PG precursors to downregulate PG synthesis in the stationary phase. Tetrapeptide accumulation relies on the substrate preference of l,d-carboxypeptidases for d-ala versus NCDAA-modified substrates. Keywords: cell wall, Vibrio cholerae, peptidoglycan recycling, l,d-carboxypeptidase, l,d-transpeptidase, NCDAA, d-amino acids, Tn-seq

A gene annotated asyiiUin the genome ofSalmonella entericaserovar Typhimurium encodes a protein homologous toE. coliZapB, a non-essential cell division factor involved in Z-ring assembly. ZapB−null mutants ofS. entericaare bile-sensitive. The ZapB protein is degraded in the presence of sodium deoxycholate (DOC), and degradation appears to involve the Lon protease. The amount ofzapBmRNA increases in the presence of a sublethal concentration of DOC. This increase is not caused by upregulation ofzapBtranscription but by increased stability ofzapBmRNA. DOC-induced increase of thezapBtranscript is suppressed by anhfqmutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. Increased stability ofzapBmRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thus providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.IMPORTANCEBile salts have bactericidal activity as a consequence of membrane disruption, protein denaturation and DNA damage. However, intestinal bacteria are resistant to bile. Envelope structures such as the lipopolysaccharide and the enterobacterial common antigen act as barriers that reduce intake of bile salts. Remodelling of the outer membrane and the peptidoglycan, activation of efflux pumps, and upregulation of stress responses also contribute to bile resistance. This study adds the cell division factor ZapB (and presumably the Z-ring) to the list of cellular functions involved in bile resistance.

When nutrients run out, bacteria enter a dormant metabolic state. This low or undetectable metabolic activity helps bacteria to preserve their scant reserves for future, but also diminishes their ability to trace the environment for new growth-promoting substrates. However, neighboring microbial growth is a sure indicator of favorable environment and thus, can serve as a cue for exiting the dormancy. Here we report that forEscherichia colithis cue is the basic peptidoglycan unit (i.e. muropeptide). We show that several forms of muropeptides can stimulate growth resumption of dormantE. colicells, but the sugar – peptide bond is crucial for activity. We also demonstrate that muropeptides from several different species can induce growth resumption ofE. coliand alsoPseudomonas aeruginosa. These results, together with the previous identification of muropeptides as germination signal for bacterial spores, makes muropeptides rather universal cue for bacterial growth.

Peptidoglycan (PG) is an essential molecule for the survival of bacteria, and thus, its biosynthesis and remodeling have always been in the spotlight when it comes to the development of antibiotics. The peptidoglycan polymer provides a protective function in bacteria, but at the same time is continuously subjected to editing activities that in some cases lead to the release of peptidoglycan fragments (i.e., muropeptides) to the environment. Several soluble muropeptides have been reported to work as signaling molecules. In this review, we summarize the mechanisms involved in muropeptide release (PG breakdown and PG recycling) and describe the known PG-receptor proteins responsible for PG sensing. Furthermore, we overview the role of muropeptides as signaling molecules, focusing on the microbial responses and their functions in the host beyond their immunostimulatory activity.

Enzymes catalysing the stereo-chemical inter-conversion of amino acids are known as amino acid racemases. In bacteria, these enzymes are fundamental to synthesize the D-Ala and D-Glu that are critical components of the peptidoglycan. In addition to this structural function in cell wall assembly, D-amino acids produced by microbial amino acid racemases have been described as relevant constituents in other prokaryotic structures (e.g. capsule, non-ribosomal peptides) and have been associated to growth fitness and to processes such as biofilm development, spore germination and signalling. The recent discovery of broad spectrum racemases able to produce and release several D-amino acids to the environment suggests that these enzymes might have a great impact in microbial ecology. Consequently, new data on the biochemistry and regulation of racemases is key to understand the biological significance of D-enantiomers in nature, in particular their effect on microbial social networks. This review summarizes current knowledge on the environmental roles of bacterial racemases with an emphasis on the potential roles of the new broad spectrum enzymes in natural environments.

Changes in the peptidoglycan (PG) structure of Salmonella enterica are detected in the presence of a sublethal concentration of sodium deoxycholate (DOC): (i) lower proportions of Braun lipoprotein ...

In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila , outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d , d -carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila . In a heterologous assembly model in E. coli , expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvAB OFF and opvAB ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB OFF subpopulation is killed and the opvAB ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only., Author Summary A tradeoff can increase the adaptive capacity of an organism at the expense of lowering the fitness conferred by specific traits. This study describes a tradeoff that confers bacteriophage resistance in Salmonella enterica at the expense of reducing its pathogenic capacity. Phase variation of the opvAB operon creates two subpopulations of bacterial cells, each with a distinct lipopolysaccharide structure. One subpopulation is large and virulent but sensitive to phages that use the lipopolysaccharide O-antigen as receptor, while the other is small and avirulent but phage resistant. In the presence of a phage that targets the O-antigen, only the avirulent subpopulation survives. However, phase variation permits resuscitation of the virulent opvAB OFF subpopulation as soon as phage challenge ceases. This transient tradeoff may illustrate the adaptive value of epigenetic mechanisms that generate bacterial subpopulations in a reversible manner.

The antibacterial activities of bile salts include detergent activity on cell membranes, protein denaturation, and DNA damage. However, many bacterial species are resistant to the antibacterial activity of bile salts. Bile salts may thus be viewed both as antibacterial compounds and as signals used by bacteria to identify bile-containing animal environments. This chapter reviews the mechanisms employed by Escherichia coli, Salmonella enterica, and other intestinal bacteria to respond to bile salts, with emphasis on their relevance for pathogenesis. The reader is also referred to two comprehensive, insightful reviews on bacterial responses to bile. Unconjugated forms of bile salts are amphipatic molecules that can enter the bacterial cell by crossing membrane bilayers. Furthermore, in the outer membrane of gram-negative bacteria, porins provide passage to both unconjugated and conjugated bile salts. Bile salts, especially if unconjugated, may easily reach the bacterial ctyoplasm by diffusion. Bile salts may also bind to specific receptors in the bacterial surface, activating signal transduction pathways that change gene expression patterns. Bile extract or individual bile salts can be added to microbiological media, thus permitting reductionist studies of bacterial bile resistance in the laboratory. It is hypothesized that nonmutational, reversible resistance may involve stochastic activation of one or more efflux systems that transport bile outside the cell.

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations., Author Summary This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile: adaptation, mutation, and non-mutational preadaptation. Adaptation is easily observed in the laboratory: when a Salmonella culture is grown in the presence of a sublethal concentration of the bile salt sodium deoxycholate (DOC), the minimal inhibitory concentration of DOC increases. Adaptation appears to be associated to multiple changes in gene expression induced by DOC. Mutational bile resistance is also a common phenomenon: plating on agar containing a lethal concentration of bile yields bile-resistant colonies. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. Non-mutational preadaptation, a non-canonical phenomenon a priori, suggests that batch cultures contain rare Salmonella cells whose physiological state permits survival upon encountering bile. The view that non-mutational preadaptation may be a consequence of phenotypic heterogeneity is supported by the observation that Salmonella cultures contain cells that activate stress response genes in the absence of DOC.

A genetic screen for suppressors of bile sensitivity in DNA adenine methylase ( dam ) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA , asmA dam , asmA phoP , and asmA wec strains of S. enterica , suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB -controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of ∼70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA + and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

Soon after birth the mammalian gut microbiota forms a permanent and collectively highly resilient consortium. There is currently no robust method for re-deriving an already microbially colonized individual again-germ-free. We previously developed the in vivo growth-incompetent E. coli K-12 strain HA107 that is auxotrophic for the peptidoglycan components D-alanine (D-Ala) and meso-diaminopimelic acid (Dap) and can be used to transiently associate germ-free animals with live bacteria, without permanent loss of germ-free status. Here we describe the translation of this experimental model from the laboratory-adapted E. coli K-12 prototype to the better gut-adapted commensal strain E. coli HS. In this genetic background it was necessary to complete the D-Ala auxotrophy phenotype by additional knockout of the hypothetical third alanine racemase metC. Cells of the resulting fully auxotrophic strain assembled a peptidoglycan cell wall of normal composition, as long as provided with D-Ala and Dap in the medium, but could not proliferate a single time after D-Ala/Dap removal. Yet, unsupplemented bacteria remained active and were able to complete their cell cycle with fully sustained motility until immediately before autolytic death. Also in vivo, the transiently colonizing bacteria retained their ability to stimulate a live-bacteria-specific intestinal Immunoglobulin (Ig)A response. Full D-Ala auxotrophy enabled rapid recovery to again-germ-free status. E. coli HS has emerged from human studies and genomic analyses as a paradigm of benign intestinal commensal E. coli strains. Its reversibly colonizing derivative may provide a versatile research tool for mucosal bacterial conditioning or compound delivery without permanent colonization. ISSN:1932-6203

When nutrients run out, bacteria enter a dormant metabolic state. This low or undetectable metabolic activity helps bacteria to preserve their scant reserves for the future needs, yet it also diminishes their ability to scan the environment for new growth-promoting substrates. However, neighboring microbial growth is a reliable indicator of a favorable environment and can thus serve as a cue for exiting dormancy. Here we report that for Escherichia coli and Pseudomonas aeruginosa this cue is provided by the basic peptidoglycan unit (i.e. muropeptide). We show that several forms of muropeptides from a variety of bacterial species can stimulate growth resumption of dormant cells and the sugar – peptide bond is crucial for this activity. These results, together with previous research that identifies muropeptides as a germination signal for bacterial spores, and their detection by mammalian immune cells, show that muropeptides are a universal cue for bacterial growth.

The electric activity of lower motor neurons (MNs) appears to play a role in determining cell-vulnerability in MN diseases. MN excitability is modulated by cholinergic inputs through C-type synaptic boutons, which display an endoplasmic reticulum-related subsurface cistern (SSC) adjacent to the postsynaptic membrane. Besides cholinergic molecules, a constellation of proteins involved in different signal-transduction pathways are clustered at C-type synaptic sites (M2 muscarinic receptors, Kv2.1 potassium channels, Ca2+ activated K+ [SK] channels, and sigma-1 receptors [S1R]), but their collective functional significance so far remains unknown. We have previously suggested that neuregulin-1 (NRG1)/ErbBs-based retrograde signalling occurs at this synapse. To better understand signalling through C-boutons, we performed an analysis of the distribution of C-bouton-associated signalling proteins. We show that within SSC, S1R, Kv2.1 and NRG1 are clustered in highly specific, non-overlapping, microdomains, whereas ErbB2 and ErbB4 are present in the adjacent presynaptic compartment. This organization may define highly ordered and spatially restricted sites for different signal-transduction pathways. SSC associated proteins are disrupted in axotomised MNs together with the activation of microglia, which display a positive chemotactism to C-bouton sites. This indicates that C-bouton associated molecules are also involved in neuroinflammatory signalling in diseased MNs, emerging as new potential therapeutic targets. We would like to thank Dr. Ronald W. Oppenheim for his critical reading of the manuscript and for helpful comments and suggestions. We would also like to thank Marta Hereu for her technical assistance, Lidia Delgado, Gema Marta Martínez and M. Yolanda Muela, from the Unitat de Criomicroscòpia Electrònica (Centres Científics i Tecnològics de la Universitat de Barcelona), for their technical support with ultrastructural immunolabelling, Daniel Cabezas for his help in some experiments, and the SCT animal facility of the Universitat de Lleida for mouse care and housing. This work was supported by grants from the Ministerio de Economía y Competitividad cofinanced by FEDER (SAF2015–70801-R to J.E.E. and J.C.), and from Jack Van den Hoek a la investigació de l’ELA - Fundació Miquel Valls (to J.E.E.).