Bacterial Approaches for Assembling Iron-Sulfur Proteins.

Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.

Making iron-sulfur cluster: structure, regulation and evolution of the bacterial ISC system.

Iron sulfur (Fe-S) clusters rank among the most ancient and conserved prosthetic groups. Fe-S clusters containing proteins are present in most, if not all, organisms. Fe-S clusters containing proteins are involved in a wide range of cellular processes, from gene regulation to central metabolism, via gene expression, RNA modification or bioenergetics. Fe-S clusters are built by biogenesis machineries conserved throughout both prokaryotes and eukaryotes. We focus mostly on bacterial ISC machinery, but not exclusively, as we refer to eukaryotic ISC system when it brings significant complementary information. Besides covering the structural and regulatory aspects of Fe-S biogenesis, this review aims to highlight Fe-S biogenesis facets remaining matters of discussion, such as the role of frataxin, or the link between fatty acid metabolism and Fe-S homeostasis. Last, we discuss recent advances on strategies used by different species to make and use Fe-S clusters in changing redox environmental conditions.

SpdC, a novel virulence factor, controls histidine kinase activity in Staphylococcus aureus.

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.

Structure and function of the Leptospira interrogans peroxide stress regulator (PerR), an atypical PerR devoid of a structural metal-binding site.

Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by H2O2 leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete Leptospira interrogans, a rare example of PerR lacking a structural metal-binding site. In vivo studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a perR mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion. In vitro, leptospiral PerR could bind to the perR promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

Various checkpoints prevent the synthesis of Staphylococcus aureus peptidoglycan hydrolase LytM in the stationary growth phase.

In Staphylococcus aureus, peptidoglycan metabolism plays a role in the host inflammatory response and pathogenesis. Transcription of the peptidoglycan hydrolases is activated by the essential 2-component system WalKR at low cell density. During stationary growth phase, WalKR is not active and transcription of the peptidoglycan hydrolase genes is repressed. In this work, we studied regulation of expression of the glycylglycine endopeptidase LytM. We show that, in addition to the transcriptional regulation mediated by WalKR, the synthesis of LytM is negatively controlled by a unique mechanism at the stationary growth phase. We have identified 2 different mRNAs encoding lytM, which vary in the length of their 5' untranslated (5'UTR) regions. LytM is predominantly produced from the WalKR-regulated mRNA transcript carrying a short 5'UTR. The lytM mRNA is also transcribed as part of a polycistronic operon with the upstream SA0264 gene and is constitutively expressed. Although SA0264 protein can be synthesized from the longer operon transcript, lytM cannot be translated because its ribosome-binding site is sequestered into a translationally inactive secondary structure. In addition, the effector of the agr system, RNAIII, can inhibit translation of lytM present on the operon without altering the transcript level but does not have an effect on the translation of the upstream gene. We propose that this dual regulation of lytM expression, at the transcriptional and post-transcriptional levels, contributes to prevent cell wall damage during the stationary phase of growth.

Single cell stochastic regulation of pilus phase variation by an attenuation-like mechanism.

The molecular triggers leading to virulence of a number of human-adapted commensal bacteria such as Streptococcus gallolyticus are largely unknown. This opportunistic pathogen is responsible for endocarditis in the elderly and associated with colorectal cancer. Colonization of damaged host tissues with exposed collagen, such as cardiac valves and pre-cancerous polyps, is mediated by appendages referred to as Pil1 pili. Populations of S. gallolyticus are heterogeneous with the majority of cells weakly piliated while a smaller fraction is hyper piliated. We provide genetic evidences that heterogeneous pil1 expression depends on a phase variation mechanism involving addition/deletion of GCAGA repeats that modifies the length of an upstream leader peptide. Synthesis of longer leader peptides potentiates the transcription of the pil1 genes through ribosome-induced destabilization of a premature stem-loop transcription terminator. This study describes, at the molecular level, a new regulatory mechanism combining phase variation in a leader peptide-encoding gene and transcription attenuation. This simple and robust mechanism controls a stochastic heterogeneous pilus expression, which is important for evading the host immune system while ensuring optimal tissue colonization.

Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system.

BACKGROUND: The Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. CONCLUSIONS/SIGNIFICANCE: A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player.

The WalKR system controls major staphylococcal virulence genes and is involved in triggering the host inflammatory response.

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 (WalR(c), 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 WalR(c) 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.

Rga, a RofA-like regulator, is the major transcriptional activator of the PI-2a pilus in Streptococcus agalactiae.

Rapid adaptation to changing environments is key in determining the outcome of infections caused by the opportunistic human pathogen Streptococcus agalactiae. We previously demonstrated that the RofA-like protein (RALP) regulators RogB and Rga activate their downstream divergently transcribed genes, that is, the pilus operon PI-2a and the serine-rich repeat encoding gene srr1, respectively. Characterization of the Rga regulon by microarray revealed that the PI-2a pilus was strongly controlled by Rga, a result confirmed at the protein level. Complementation experiments showed that the expression of Rga, but not RogB, in the double ΔrogB/Δrga mutant, or in the clinical strain 2603V/R displaying frameshift mutations in rogB and rga genes, is sufficient to restore wild-type expression levels of PI-2a pilus and Srr1. Biofilm formation was impaired in the Δrga and Δrga/rogB mutants and restored on complementation with rga. Paradoxically, adherence to intestinal epithelial cells was unchanged in the Δrga mutant. Finally, the existence of several clinical isolates mutated in rga highlights the concept of strain-specific regulatory networks.

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 pleiotropic CymR regulator of Staphylococcus aureus plays an important role in virulence and stress response.

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a DeltacymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the DeltacymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the DeltacymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host.

Novel antibacterial compounds specifically targeting the essential WalR response regulator.

The WalK/WalR (YycG/YycF) two-component system, which is essential for cell viability, is highly conserved and specific to low-GC percentage of Gram-positive bacteria, making it an attractive target for novel antimicrobial compounds. Recent work has shown that WalK/WalR exerts an effect as a master regulatory system in controlling and coordinating cell wall metabolism with cell division in Bacillus subtilis and Staphylococcus aureus. In this paper, we develop a high-throughput screening system for WalR inhibitors and identify two novel inhibitors targeting the WalR response regulator (RR): walrycin A (4-methoxy-1-naphthol) and walrycin B (1,6-dimethyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4-e][1,2,4]triazine-5,7-dione). Addition of these compounds simultaneously affects the expression of WalR regulon genes, leading to phenotypes consistent with those of cells starved for the WalK/WalR system and having a bactericidal effect. B. subtilis cells form extremely long aseptate filaments and S. aureus cells form large aggregates under these conditions. These results show that walrycins A and B are the first antibacterial agents targeting WalR in B. subtilis and S. aureus.

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.

The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis.

DegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU-dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU(D55N)) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU(D55N) mutant. We also show that the small-molecular-weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesDeltaptaDeltaackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes.

Tearing down the wall: peptidoglycan metabolism and the WalK/WalR (YycG/YycF) essential two-component system.

In order to survive, bacteria have developed avariety of highly sophisticated and sensitive signal transduction pathways with which they adapt their genetic expression to meet the challenges of their ever-changing surroundings. These mechanisms enable bacterial cells to communicate with their environment, their hosts and each other, allowing them adopt specific responses, or develop specialised structures such as biofilms or spores to ensure survival, colonization of their ecological niches and dissemination. As highlighted in this book, the so-called two-component systems (TCSs) are one of the most widespread and efficient strategies used for this purpose, where signal acquisition involves autophosphorylation of a sensor histidine kinase and transduction takes place when the kinase phosphorylates its cognate response regulator protein, leading in turn to specific alteration ofgene expression. In their simplest form, TCSs elegantly combine sensing, transducing and transcription activation modules within two proteins, effectively coupling external signals to genetic adaptation. The high degree of conservation among TCS phosphotransfer domains, their ubiquitous nature and the fact that several are essential for cell viability has made them an attractive target for novel classes of antimicrobial compounds. The WalK/WalR (aka YycG/YycF) two-component system, originally identified in Bacillus subtilis, is very highly conserved and specific to low G + C Gram-positive bacteria, including several pathogens such as Staphylococcus aureus. While this system is essential for cell viability, both the nature of its regulon and its physiological role had remained mostly uncharacterized. A number of recent studies have now unveiled a conserved function for this system in different bacteria, defining this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR. This review will focus on the cellular function of the WalK/WalR TCS in different bacterial species and the attractive target it constitutes for novel classes of antimicrobial compounds.

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.

The Listeria monocytogenes virulence factor InlJ is specifically expressed in vivo and behaves as an adhesin.

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.