Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS‐DegU two‐component system. Here we report a role for flagella in the regulation of the K‐state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU‐P, which inhibits the expression of ComK, the master regulator for the K‐state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hag^A233V) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU‐P levels through an unknown signaling mechanism. This flagellar‐load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K‐state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.     

Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.     

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate

Bacteria undergoing nutrient starvation induce the ubiquitous stringent response, resulting in gross physiological changes that reprograms cell metabolism from fast to slow growth. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or ‘alarmone’. In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation, whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that FA starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNA^Lys and activation of RelA. SpoT was also activated by FA starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results raise the possibility that RelA may be a major player under many starvation conditions previously thought to depend principally on SpoT. Interestingly, FA starvation provoked a ~100‐fold increase in relA dependent ampicillin tolerance.     

HipA-Triggered Growth Arrest and β-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis

Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.     

Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two‐component systems in phosphate‐limited Bacillus subtilis cells

In Bacillus subtilis, the WalRK (YycFG) two‐component system controls peptidoglycan metabolism in exponentially growing cells while PhoPR controls the response to phosphate limitation. Here we examine the roles of WalRK and PhoPR in peptidoglycan metabolism in phosphate‐limited cells. We show that B. subtilis cells remain viable in a phosphate‐limited state for an extended period and resume growth rapidly upon phosphate addition, even in the absence of a PhoPR‐mediated response. Peptidoglycan synthesis occurs in phosphate‐limited wild‐type cells at ～27% the rate of exponentially growing cells, and at ～18% the rate of exponentially growing cells in the absence of PhoPR. In phosphate‐limited cells, the WalRK regulon genes yocH, cwlO(yvcE), lytE and ydjM are expressed in a manner that is dependent on the WalR recognition sequence and deleting these genes individually reduces the rate of peptidoglycan synthesis. We show that ydjM expression can be activated by PhoP～P in vitro and that PhoP occupies its promoter in phosphate‐limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP～P in vitro, but can be repressed by non‐phosphorylated WalR in vitro. Therefore, we conclude that peptidoglycan metabolism is controlled by both WalRK and PhoPR in phosphate‐limited B. subtilis cells.     

PfeT,a P1B4‐type ATPase,effluxes ferrous iron and protects Bacillus subtilis against iron intoxication

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P_1B4‐type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild‐type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini‐ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P_1B4‐type ATPases previously implicated in bacterial pathogenesis.     

Exploring ComQXPA quorum‐sensing diversity and biocontrol potential of Bacillus spp. isolates from tomato rhizoplane

Bacillus subtilis is a widespread and diverse bacterium t exhibits a remarkable intraspecific diversity of the ComQXPA quorum‐sensing (QS) system. This manifests in the existence of distinct communication groups (pherotypes) that can efficiently communicate within a group, but not between groups. Similar QS diversity was also found in other bacterial species, and its ecological and evolutionary meaning is still being explored. Here we further address the ComQXPA QS diversity among isolates from the tomato rhizoplane, a natural habitat of B. subtilis, where these bacteria likely exist in their vegetative form. Because this QS system regulates production of anti‐pathogenic and biofilm‐inducing substances such as surfactins, knowledge on cell–cell communication of this bacterium within rhizoplane is also important from the biocontrol perspective. We confirm the presence of pherotype diversity within B. subtilis strains isolated from a rhizoplane of a single plant. We also show that B. subtilis rhizoplane isolates show a remarkable diversity of surfactin production and potential plant growth promoting traits. Finally, we discover that effects of surfactin deletion on biofilm formation can be strain specific and unexpected in the light of current knowledge on its role it this process.     

Relacin,a Novel Antibacterial Agent Targeting the Stringent Response

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.     

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.