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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.

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan puri- fied from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropep- tides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)- meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This pepti- doglycan has a reduced glycan chain average length and ~30% increase in the L,D-cross- link, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D- transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L, D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhi- murium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remark- ably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-con- taining muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D- alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells., This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.). S.C. was recipient of an EMBO Short-Term Fellowship number 6426 for a stay in the lab of F.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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.

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

The hyperproduction of the chromosomal AmpC beta-lactamase is the main mechanism driving beta-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-Nacetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of beta-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent beta-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling. IMPORTANCE The extensive use of beta-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC beta-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance., Work in the Oliver lab is supported by the Sociedad Espanola de Enfermedades Infecciosas y Microbiologia Clinica (SEIMC), the Ministerio de Economia y Competitividad of Spain, and the Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF; A way to achieve Europe) through the Spanish Network for the Research in Infectious Diseases (RD12/0015 and RD16/0016), and grants CP12/03324, PI15/00088, PI15/02212, PI18/00681, and PI18/00076. Work in the Cava lab is supported by the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation (KAW), the Laboratory of Molecular Infection Medicine Sweden (MIMS), and the Kempe Foundation.

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.

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 (Lpp)-bound muropeptides; (ii) reduced levels of muropeptides cross-linked by L(meso)-diaminopimelyl-D(meso)-diaminopimelic acid (L-D) peptide bridges (3-3 cross-links). Similar structural changes are found in S. enterica cultures adapted to grow in the presence of a lethal concentration of DOC, suggesting that reduced anchoring of Braun protein to PG and low occurrence of 3-3 cross-links may increase S. enterica resistance to bile. This view is further supported by additional observations: (i) A triple mutant lacking L,D-transpeptidases YbiS, ErfK, and YcfS, which does not contain Lpp anchored to PG, is hyper-resistant to bile; (ii) enhanced 3-3 cross-linking upon overexpression of YnhG transpeptidase causes a decrease in bile resistance. These observations suggest that remodelling of the cell wall may be added to the list of adaptive responses that permit survival of S. enterica in the presence of bile.

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.