Genetic and Biochemical Characterization of the MinC-FtsZ Interaction in Bacillus subtilis

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.     

Prediction of the Mechanism of Action of Fusaricidin on Bacillus subtilis

Long-term use of antibiotics has engendered a large number of resistant pathogens, which pose a serious threat to human health. Here, we investigated the mechanism of fusaricidin antibacterial activity toward Bacillus subtilis and characterized the pathways responsible for drug resistance. We found that σ^w, an extracytoplasmic function sigma factor, plays an important role in the resistance to fusaricidins during the initial 5 minutes of drug addition. Approximately 18 genes were induced more than 3-fold, of which 66.7% are known to be regulated by σ^w. Over the following 3 h, fusaricidins induced 194 genes more than three-fold, and most were associated with classes of antibiotic-responsive stimulons. Moreover, the fusaricidin treatment increased the catabolism of fatty and amino acids but strongly repressed glucose decomposition and gluconeogenesis. In summary, our data provide insight into the mechanism of fusaricidin activity, on which we based our suggested strategies for the development of novel antibiotic agents.     

MreB-Dependent Inhibition of Cell Elongation during the Escape from Competence in Bacillus subtilis

During bacterial exponential growth, the morphogenetic actin-like MreB proteins form membrane-associated assemblies that move processively following trajectories perpendicular to the long axis of the cell. Such MreB structures are thought to scaffold and restrict the movement of peptidoglycan synthesizing machineries, thereby coordinating sidewall elongation. In Bacillus subtilis, this function is performed by the redundant action of three MreB isoforms, namely MreB, Mbl and MreBH. mreB and mbl are highly transcribed from vegetative promoters. We have found that their expression is maximal at the end of exponential phase, and rapidly decreases to a low basal level upon entering stationary phase. However, in cells developing genetic competence, a stationary phase physiological adaptation, expression of mreB was specifically reactivated by the central competence regulator ComK. In competent cells, MreB was found in complex with several competence proteins by in vitro pull-down assays. In addition, it co-localized with the polar clusters formed by the late competence peripheral protein ComGA, in a ComGA-dependent manner. ComGA has been shown to be essential for the inhibition of cell elongation characteristic of cells escaping the competence state. We show here that the pathway controlling this elongation inhibition also involves MreB. Our findings suggest that ComGA sequesters MreB to prevent cell elongation and therefore the escape from competence.     

Menaquinone and Iron Are Essential for Complex Colony Development in Bacillus subtilis

Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD) in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis.     

One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.     

Modeling Curvature-Dependent Subcellular Localization of the Small Sporulation Protein SpoVM in Bacillus subtilis

Recent in vivo experiments suggest that in the bacterium, Bacillus subtilis, the cue for the localization of the small sporulation protein, SpoVM, an essential factor in spore coat formation, is curvature of the bacterial plasma membrane. In vitro measurements of SpoVM adsorption to vesicles of varying sizes also find high sensitivity of adsorption to vesicle radius. This curvature-dependent adsorption is puzzling given the orders of magnitude difference in length scale between an individual protein and the radius of curvature of the cell or vesicle, suggesting protein clustering on the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane adsorption and clustering of SpoVM. Based on our analysis, we hypothesize that the radius dependence of SpoVM adsorption observed in vitro is governed primarily by membrane tension, while for in-vivo localization of SpoVM, we propose a highly sensitive mechanism for curvature sensing based on the formation of macroscopic protein clusters on the membrane.     

Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.     

Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in Bacillus subtilis

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.     

Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis

In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded RNA (dsRNA) to knockdown translation of targeted mRNAs. However, it has been shown that E. coli is mildly pathogenic to C. elegans and this pathogenicity might influence aging and the accuracy of the RNAi-screening during aging may as well be affected. Here, we describe a novel system that utilizes the non-pathogenic bacterium Bacillus subtilis, to express dsRNA and therefore eliminates the effects of bacterial pathogenicity from the genetic analysis of aging.     

Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.     

Dual-Specificity Anti-sigma Factor Reinforces Control of Cell-Type Specific Gene Expression in Bacillus subtilis

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σ^Fand σ^E control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σ^F is replaced by σ^G and σ^E is replaced by σ^K. The anti-sigma factor CsfB is produced under the control of σ^F and binds to and inhibits the auto-regulatory σ^G, but not σ^F. A position in region 2.1, occupied by an asparagine in σ^G and by a glutamate in ο^F, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σ^K and that CsfB binds to and inhibits σ^E but not σ^K, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σ^E and by a conserved glutamate in σ^K suffices for discrimination by CsfB. We also show that CsfB prevents activation of σ^G in the mother cell and the premature σ^G-dependent activation of σ^K. Thus, CsfB establishes negative feedback loops that curtail the activity of σ^E and prevent the ectopic activation of σ^G in the mother cell. The capacity of CsfB to directly block σ^E activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σ^E activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σ^F/σ^G and σ^E/σ^K pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.     

High Levels of DegU-P Activate an Esat-6-Like Secretion System in Bacillus subtilis

The recently discovered Type VII/Esat-6 secretion systems seem to be widespread among bacteria of the phyla Actinobacteria and Firmicutes. In some species they play an important role in pathogenic interactions with eukaryotic hosts. Several studies have predicted that the locus yukEDCByueBC of the non-pathogenic, Gram-positive bacterium Bacillus subtilis would encode an Esat-6-like secretion system (Ess). We provide here for the first time evidences for the functioning of this secretion pathway in an undomesticated B. subtilis strain. We show that YukE, a small protein with the typical features of the secretion substrates from the WXG100 superfamily is actively secreted to culture media. YukE secretion depends on intact yukDCByueBC genes, whose products share sequence or structural homology with known components of the S. aureus Ess. Biochemical characterization of YukE indicates that it exists as a dimer both in vitro and in vivo. We also show that the B. subtilis Ess essentially operates in late stationary growth phase in absolute dependence of phosphorylated DegU, the response regulator of the two-component system DegS-DegU. We present possible reasons that eventually have precluded the study of this secretion system in the B. subtilis laboratory strain 168.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.     

The Eukaryotic-Like Ser/Thr Kinase PrkC Regulates the Essential WalRK Two-Component System in Bacillus subtilis

Most bacteria contain both eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs). Their role in bacterial physiology is not currently well understood in large part because the conditions where the eSTKs are active are generally not known. However, all sequenced Gram-positive bacteria have a highly conserved eSTK with extracellular PASTA repeats that bind cell wall derived muropeptides. Here, we report that in the Gram-positive bacterium Bacillus subtilis, the PASTA-containing eSTK PrkC and its cognate eSTP PrpC converge with the essential WalRK two-component system to regulate WalR regulon genes involved in cell wall metabolism. By continuously monitoring gene expression throughout growth, we consistently find a large PrkC-dependent effect on expression of several different WalR regulon genes in early stationary phase, including both those that are activated by WalR (yocH) as well as those that are repressed (iseA, pdaC). We demonstrate that PrkC phosphorylates WalR in vitro and in vivo on a single Thr residue located in the receiver domain. Although the phosphorylated region of the receiver domain is highly conserved among several B. subtilis response regulators, PrkC displays specificity for WalR in vitro. Consistently, strains expressing a nonphosphorylatable WalR point mutant strongly reduce both PrkC dependent activation and repression of yocH, iseA, and pdaC. This suggests a model where the eSTK PrkC regulates the essential WalRK two-component signaling system by direct phosphorylation of WalR Thr101, resulting in the regulation of WalR regulon genes involved in cell wall metabolism in stationary phase. As both the eSTK PrkC and the essential WalRK two-component system are highly conserved in Gram-positive bacteria, these results may be applicable to further understanding the role of eSTKs in Gram-positive physiology and cell wall metabolism.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.