Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. , the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogenVibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogensNeisseria gonorrheaeandEscherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Bacteria encode a variety of adaptations that enable them to survive during zinc starvation, a condition which is encountered both in natural environments and inside the human host. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a novel member of this zinc starvation response, a cell wall hydrolase that retains function and is conditionally essential for cell growth in low-zinc environments. Other Gram-negative bacteria contain homologs that appear to be under similar regulatory control. These findings are significant because they represent, to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective therapies commonly target the bacterial cell wall; therefore, an improved understanding of how the cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such therapeutic interventions are required to combat the rising threat of drug-resistant infections.

17The cell wall is a strong, yet flexible, meshwork of peptidoglycan (PG) that gives a bacterium structural 18 integrity. To accommodate a growing cell, the wall is remodeled by both PG synthesis and degradation. 19 Vibrio cholerae encodes a group of three nearly identical zinc-dependent endopeptidases (EPs) that 20 hydrolyze PG to facilitate cell growth. Two of these (shyA and shyC) are housekeeping genes and form a 21 synthetic lethal pair, while the third (shyB) is not expressed under standard laboratory conditions. To 22 investigate the role of ShyB, we conducted a transposon screen to identify mutations that activate shyB 23 transcription. We found that shyB is induced as part of the Zur-mediated zinc starvation response, a 24 mode of regulation not previously reported for cell wall lytic enzymes. In vivo, ShyB alone was sufficient to sustain cell growth in low-zinc environments. In vitro, ShyB retained its D,D-endopeptidase 26 activity against purified sacculi in the presence of the metal chelator EDTA at a concentration that 27 inhibits ShyA and ShyC. This suggests that ShyB can substitute for the other EPs during zinc starvation, 28 a condition that pathogens encounter while infecting a human host. Our survey of transcriptomic data 29 from diverse bacteria identified other candidate Zur-regulated endopeptidases, suggesting that this 30 adaptation to zinc starvation is conserved in other Gram-negative bacteria. 31 32 Importance 33The human host sequesters zinc and other essential metals in order to restrict growth of potentially 34 harmful bacteria. In response, invading bacteria express a set of genes enabling them to cope with zinc 35 starvation. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a 36 novel member of this zinc starvation response: a cell wall hydrolase that retains function in low-zinc 37 environments and is conditionally essential for cell growth. Other human pathogens contain homologs 38 that appear to be under similar regulatory control. These findings are significant because they represent, 39 to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective 40 therapies commonly target the bacterial cell wall and, therefore, an improved understanding of how the 41 cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such 42 therapeutic interventions are required to combat the rising threat of drug resistant infections. 43 44 strands, enabling the PG to assemble into a meshlike structure called the sacculus (3). In Gram-negative 51 bacteria, the sacculus is a single PG layer that is sandwiched between an inner and an outer membrane 52 (4). This thin wall must be rigid enough to maintain cell shape and to contain high intracellular pressures 53 (3, 5). However, the wall must also be flexible enough to accommodate cell elongation, cell division, 54 and the insertion of trans-envelope protein complexes (6). This requirement for both rigidity and 55 flexibility ...

Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae’s life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. cholerae’s zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc starvation genes (Δzur) congregates at the bottom of a culture tube when grown in a nutrient-poor medium. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes were required for congregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and an activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent congregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and energy taxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.