Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.Organismic and Evolutionary Biolog

Isentropic Compression Experiments Performed By LLNL On Energetic Material Samples Using The Z Accelerator

Several experiments have been conducted by LLNL researchers using isentropic compression experiments (ICE) on energetic materials as samples from Fiscal Year 2001 (FY01) to Fiscal Year 2005 (FY05). Over this span of time, advancements of the experimental techniques and modeling of the results have evolved to produce improved results. This report documents the experiments that have been performed, provides details of the results generated, and modeling and analysis advances to fully understand the results. Publications on the topics by the various principal investigators (PI's) are detailed in the Appendices for quick reference for the work as it progressed

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

ABSTRACTInitiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies

Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKRB, formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKRG, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers