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Bacterial cells are encased in a peptidoglycan (PG) exoskeleton that protects them from osmotic lysis and specifies their distinct shapes. Cell wall hydrolases are required to enlarge this covalently closed macromolecule during growth, but how these autolytic enzymes are regulated remains poorly understood. Bacillus subtilis encodes two functionally redundant D,L-endopeptidases (CwlO and LytE) that cleave peptide crosslinks to allow expansion of the PG meshwork during growth. Here, we provide evidence that the essential and broadly conserved WalR-WalK two component regulatory system continuously monitors changes in the activity of these hydrolases by sensing the cleavage products generated by these enzymes and modulating their levels and activity in response. The WalR-WalK pathway is conserved among many Gram-positive pathogens where it controls transcription of distinct sets of PG hydrolases. Cell wall remodeling in these bacteria may be subject to homeostatic control mechanisms similar to the one reported here.

Penicillin and related antibiotics disrupt cell wall synthesis in bacteria causing the downstream misactivation of cell wall hydrolases called autolysins to induce cell lysis. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae ( Sp ), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked teichoic acids (LTAs). Because LytA binds to both species of teichoic acids, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis during exponential growth . We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active peptidoglycan (PG) synthesis. Our results support a model in which the WTA tailoring activity of WhyD during exponential growth directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens.

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica . It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers. The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers.

Most bacterial cells are surrounded by an essential cell wall composed of the net-like heteropolymer peptidoglycan (PG). Growth and division of bacteria are intimately linked to the expansion of the PG meshwork and the construction of a cell wall septum that separates the nascent daughter cells. Class A penicillin-binding proteins (aPBPs) are a major family of PG synthases that build the wall matrix. Given their central role in cell wall assembly and importance as drug targets, surprisingly little is known about howthe activity of aPBPs is controlled to properly coordinate cell growth and division. Here, we report the identification of MacP (SPD_0876) as a membrane-anchored cofactor of PBP2a, an aPBP synthase of the Gram-positive pathogen Streptococcus pneumoniae. We show that MacP localizes to the division site of S. pneumoniae, forms a complex with PBP2a, and is required for the in vivo activity of the synthase. Importantly, MacP was also found to be a substrate for the kinase StkP, a global cell cycle regulator. Although StkP has been implicated in controlling the balance between the elongation and septation modes of cell wall synthesis, none of its substrates are known to modulate PG synthetic activity. Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP–PBP2a interaction. Our results thus reveal a direct connection between PG synthase function and the control of cell morphogenesis by the StkP regulatory network.

The cell envelope of pathogenic bacteria is a barrier against host environmental conditions and immunity molecules, as well as the site where many virulence factors are assembled. Extracytoplasmic stress responses (ESRs) have evolved to help maintain its integrity in conditions where it might be compromised. These ESRs also have important links to the production of envelope-associated virulence systems by the bacteria themselves. One such virulence factor is the type III secretion system (T3SS), the first example of which was provided by the pathogenic Yersinia. This article reviews the reported links between four different ESRs and T3SS function in Yersinia. Components of three of these ESRs affect the function and/or regulation of two different T3SSs. The response regulator of the Rcs ESR is involved in positive regulation of the Ysa-Ysp T3SS found in the highly pathogenic 1B biogroup of Y. enterocolitica. Conversely, the response regulator of the Y. pseudotuberculosis Cpx ESR can down-regulate production of the Ysc-Yop T3SS, and at least one other envelope virulence factor (invasin), by direct repression. Also in Y. pseudotuberculosis, there is some evidence suggesting that an intact RpoE ESR might be important for normal Yersinia outer proteins (Yop) production and secretion. Besides these regulatory links between ESRs and T3SSs, perhaps the most striking connection between T3SS function and an ESR is that between the phage shock protein (Psp) and Ysc-Yop systems of Y. enterocolitica. The Psp response does not affect the regulation or function of the Ysc-Yop system. Instead, Ysc-Yop T3SS production induces the Psp system, which then mitigates T3SS-induced envelope stress. Consequently, the Y. enterocolitica Psp system is essential when the Ysc-Yop T3SS is produced.

Multi-drug resistant Gram-negative bacteria, including carbapenem-resistant (CR ), are a public health emergency. The predominant CR sequence type worldwide is ST258. However, the factors underlying ST258's epidemic success are not well defined. The understudied two-component system CrrAB is a genomic feature of ST258 and has been hypothesized to contribute to its global dominance. Despite this, the molecular details underpinning CrrAB's contribution to ST258 pathogenicity are not well understood. We used RNA-sequencing to identify the regulon of CrrA and found that CrrAB induces the expression of a novel gene, encoding Crr-regulated fimbriae modifying protein (CfmP). CfmP post-translationally modifies fimbriae to significantly increase host cell adhesion and high bacterial loads within the host, consequently increasing ST258 virulence. CrrAB also drives high antibiotic resistance in CR . Thus, our data places CrrAB at the intersection of virulence and antibiotic resistance supporting its function as an important regulatory system driving the pathogenicity of ST258.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae. Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse–chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Penicillin and related antibiotics disrupt cell wall synthesis in bacteria and induce lysis by misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

The Phage-shock-protein (Psp) response is a mechanism used by bacteria to counteract cell envelope stress. Discovered in E. coli upon phage infection, and widely conserved among Gram-negative bacteria, the Psp response is essential for the virulence of the gastrointestinal pathogen Yersinia enterocolitica. This is because it prevents lethal envelope stress that can be caused by mislocalized \"secretin\" proteins, which are critical for virulence factor export. Communication between integral (PspB and -C) and peripheral (PspA) cytoplasmic membrane proteins is essential for activation of the Psp response. The integral membrane location of PspB and -C is consistent with a hypothesis that one or both of them detects an inducing signal and transduces it to the cytoplasm by interacting with PspA. However, the molecular details of the signal transduction mechanism between PspBC and PspA were largely unknown. To address this, I began by investigating if changes in two fundamental features of PspB and -C might occur as part of the inducing mechanism: 1) Their multimerization status; and 2) Their membrane topology. I detected PspB and -C homo- and heterodimers, but they were present regardless of the activation status of the Psp response. In addition, the membrane topologies of PspB and -C did not change upon induction. However, a major surprise was my discovery of a previously unrecognized polytopic membrane arrangement for PspC. This led to a new regulatory model founded upon the hypothesis that the availability of a PspA-binding site within the C-terminal domain of PspC is the molecular mechanism of signal transduction from membrane to cytoplasm. In vitro and in vivo protein-protein interaction studies, along with other in vivo experiments, supported this model. Importantly, my work has also provided data supporting an unforeseen negative regulatory role for PspB, which might be achieved by tethering the C-terminal domain of PspC in non-inducing conditions, perhaps to prevent PspC from sequestering PspA.