Sarah Dubrac

New Insights into the WalK/WalR (YycG/YycF) Essential Signal Transduction Pathway Reveal a Major Role in Controlling Cell Wall Metabolism and Biofilm Formation in Staphylococcus aureus

The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly, Staphylococcus aureus cell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two major S. aureus autolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved in S. aureus cell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example in S. aureus of a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.

A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway

The WalK/WalR (aka YycG/YycF) two‐component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and specific to low G+C Gram‐positive bacteria, including a number of important pathogens. An unusual feature is that this system is essential for viability in most of these bacteria. Recent studies have revealed conserved functions for this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism, that we have accordingly renamed WalK/WalR. Here we review the cellular role of the WalK/WalR TCS in different bacterial species, focusing on the function of genes in its regulon, as well as variations in walRK operon structure and the composition of its regulon. We also discuss the nature of its essentiality and the potential type of signal being sensed. The WalK histidine kinase of B. subtilis has been shown to localize to the divisome and we suggest that the WalKR system acts as an information conduit between extracytoplasmic cellular structures and intracellular processes required for their synthesis, playing a vital role in effectively co‐ordinating peptidoglycan plasticity with the cell division process.

Genes controlled by the essential YycG/YycF two‐component system of Bacillus subtilis revealed through a novel hybrid regulator approach

The YycG/YycF two‐component system, originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low G + C Gram‐positive bacteria. This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Using a combined hybrid regulator/transcriptome approach involving the inducible expression of a PhoP′‐′YycF chimerical protein in B. subtilis, we have shown that expression of yocH, which encodes a potential autolysin, is specifically activated by YycF. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the regulatory regions of yocH as well as ftsAZ, previously reported to be controlled by YycF. Nucleotide sequence analysis and site‐directed mutagenesis allowed us to define a potential consensus recognition sequence for the YycF response regulator, composed of two direct repeats: 5′‐TGT A/T A A/T/C‐N5‐TGT A/T A A/T/C‐3′. A DNA‐motif analysis indicates that there are potentially up to 10 genes within the B. subtilis YycG/YycF regulon, mainly involved in cell wall metabolism and membrane protein synthesis. Among these, YycF was shown to bind directly to the region upstream from the ykvT gene, encoding a potential cell wall hydrolase, and the intergenic region of the tagAB/tagDEF divergon, encoding essential components of teichoic acid biosynthesis. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in other low G + C Gram‐positive bacteria, including several pathogens such as Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.

The WalKR System Controls Major Staphylococcal Virulence Genes and Is Involved in Triggering the Host Inflammatory Response

ABSTRACT The WalKR two-component system is essential for the viability of Staphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR in S. aureus through a phosphomimetic amino acid replacement (WalRc, D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb, emp, fnbA, and fnbB), cytolysis (hlgACB, hla, and hlb), and innate immune defense evasion (scn, chp, and sbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalRc are strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively active walR c allele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

Interactions between the YycFG and PhoPR two‐component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non‐cognate YycF response regulator upon phosphate limitation

Two‐component signal transduction systems (TCS) are an important mechanism by which bacteria sense and respond to their environment. Although each two‐component system appears to detect and respond to a specific signal(s), it is now evident that they do not always act independently of each other. In this paper we present data indicating regulatory links between the PhoPR two‐component system that participates in the cellular response to phosphate limitation, and the essential YycFG two‐component system in Bacillus subtilis. We show that the PhoR sensor kinase can activate the YycF response regulator during a phosphate limitation‐induced stationary phase, and that this reaction occurs in the presence of the cognate YycG sensor kinase. Phosphorylation of YycF by PhoR also occurs in vitro, albeit at a reduced level. However, the reciprocal cross‐phosphorylation does not occur. A second level of interaction between PhoPR and YycFG is indicated by the fact that cells depleted for YycFG have a severely deficient PhoPR‐dependent phosphate limitation response and that YycF can bind directly to the promoter of the phoPR operon. YycFG‐depleted cells neither activate expression of phoA and phoPR nor repress expression of the essential tagAB and tagDEF operons upon phosphate limitation. This effect is specific to the PhoPR‐dependent phosphate limitation response because PhoPR‐independent phosphate limitation responses can be initiated in YycFG‐depleted cells.

The Listeria monocytogenes Virulence Factor InlJ Is Specifically Expressed In Vivo and Behaves as an Adhesin

ABSTRACT The food-borne pathogen Listeria monocytogenes is adapted to a diversity of environments, such as soil, food, body fluids, and the cytosol of eukaryotic cells. The transition between saprophytic and pathogenic life is mediated through complex regulatory pathways that modulate the expression of virulence factors. Here we examined the expression of inlJ, a recently identified gene encoding a protein of the LPXTG-internalin family and involved in pathogenesis. We show that inlJ expression is controlled neither by the major listerial regulator of virulence genes, PrfA, nor by AxyR, a putative AraC regulator encoded by a gene adjacent to inlJ and divergently transcribed. The InlJ protein is not produced by bacteria grown in vitro in brain heart infusion medium or replicating in the cytosol of tissue-cultured cells. In contrast, it is efficiently produced and localized at the surface of bacteria present in the liver and blood of infected animals. Strikingly, the expression of inlJ by a heterologous promoter in L. monocytogenes or L. innocua promotes bacterial adherence to human cells in vitro. Taken together, these results strongly suggest that InlJ acts as a novel L. monocytogenes sortase-anchored adhesin specifically expressed during infection in vivo.

Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two sigma(A)-dependent promoters, P(1) and P(2). Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P(1). During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP approximately P)-dependent manner; in addition to P(1), the P(2) promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP approximately P could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP approximately P to induce other members of the Pho regulon. The extent to which promoters P(1) and P(2) are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

Peptidoglycan Crosslinking Relaxation Plays an Important Role in Staphylococcus aureus WalKR-Dependent Cell Viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl–glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

The DegU orphan response regulator of Listeria monocytogenes autorepresses its own synthesis and is required for bacterial motility, virulence and biofilm formation

The Gram-positive intracellular pathogen Listeria monocytogenes is endowed with 17 sets of genes encoding two-component systems. L. monocytogenes is closely related to the Gram-positive model bacterium Bacillus subtilis, in which we have shown previously that the DegS/DegU system plays a central role in controlling stationary phase adaptive responses, including degradative enzyme synthesis and competence. Although an orthologue of the DegU response regulator is present in L. monocytogenes, the gene encoding the cognate DegS kinase is conspicuously absent. We have inactivated the degU gene of L. monocytogenes and shown that DegU negatively regulates its own synthesis. Direct binding of L. monocytogenes DegU to its own promoter region was shown in vitro by gel mobility shift and DNase I footprinting experiments. DegU was also shown to bind upstream from the motB operon, which also encodes the GmaR anti-repressor of flagellar synthesis. In contrast to the situation in B. subtilis, DegU was shown to be essential for flagellar synthesis and bacterial motility in L. monocytogenes and is cotranscribed with the yviA gene located downstream. We also show that DegU is required for growth at high temperatures, adherence to plastic surfaces and the formation of efficient biofilms by L. monocytogenes. DegU plays a role in virulence of L. monocytogenes as well: in a murine intravenous infection model, an 11-fold increase in LD(50) was observed for the degU mutant. Taken together, our results indicate that despite the lack of the DegS kinase, DegU is fully functional as an orphan response regulator, and plays a central role in controlling several crucial adaptive responses in L. monocytogenes.

Dual Role of the PhoP∼P Response Regulator: Bacillus amyloliquefaciens FZB45 Phytase Gene Transcription Is Directed by Positive and Negative Interactions with the phyC Promoter

Several Bacillus strains secrete phytase, an enzyme catalyzing dephosphorylation of myo-inositol hexakisphosphate (phytate). We identified the phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoPP is essential for phyC transcription. The transcriptional start site was identified downstream of a A -like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the phyC promoter sequence revealed an unusual structure. The 35 and 10 regions are separated by a window of 21 bp. A pair of tandemly repeated PhoP TT(T/A/C)ACA binding boxes was located within and upstream of the 35 consensus promoter region. A single PhoP box was found within the 10 consensus promoter region. DNase I footprinting experiments performed with isolated PhoP confirmed that PhoPP binds at two sites overlapping with the phyC 35 and 10 consensus promoter region. While binding of dimeric PhoP Pa t35 is essential for activation of the phyC promoter, binding of PhoP Pa t10 suppresses promoter activity. A sixfold enhancement of phyC gene expression was registered after T:G substitution of nucleotide 13 (mutant MUT13), which eliminates PhoP binding at the single PhoP box without impairing the 10 consensus sequence. Moreover, MUT13 also expressed phyC during phosphate-replete growth, suggesting that the repressing effect due to binding of PhoP Pa t10 was abolished. A model is presented in which transcription initiation of phyC is positively and negatively affected by the actual concentration of the PhoPP response regulator.