Expanding the genetic toolbox for the obligate human pathogen .

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen To overcome this limitation and enable the straightforward investigation of gene functions in , we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for .

remains translationally active after CRISPRi-mediated replication initiation arrest.

Initiation of bacterial DNA replication takes place at the origin of replication (), 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 during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium .

Time-Resolved Proteome Analysis of during Infection Reveals the Role of the AAA+ Chaperone ClpC for Host Cell Adaptation.

The cellular proteome comprises all proteins expressed at a given time and defines an organism's phenotype under specific growth conditions. The proteome is shaped and remodeled by both protein synthesis and protein degradation. Here, we developed a new method which combines metabolic and chemical isobaric peptide labeling to simultaneously determine the time-resolved protein decay and synthesis in an intracellular human pathogen.

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, SKR , formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH.

Functional Diversity of AAA+ Protease Complexes in .

Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism . We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

Identification and characterization of mutations conferring resistance to D-amino acids in Bacillus subtilis.

Unlabelled: Bacteria produce d-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However, D-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium Bacillus subtilis are particularly sensitive to the growth-inhibitory effects of D-tyrosine due to the absence of D-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation of D-tyrosine and other D-amino acids into nascent proteins.

The extracellular matrix of Staphylococcus aureus biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH.

Unlabelled: Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase.

Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase.

We report that the Bacillus subtilis exopolysaccharide (EPS) is a signaling molecule that controls its own production. EPS synthesis depends on a tyrosine kinase that consists of a membrane component (EpsA) and a kinase component (EpsB). EPS interacts with the extracellular domain of EpsA, which is a receptor, to control kinase activity.

The role of thiol oxidative stress response in heat-induced protein aggregate formation during thermotolerance in Bacillus subtilis.

Using Bacillus subtilis as a model organism, we investigated thermotolerance development by analysing cell survival and in vivo protein aggregate formation in severely heat-shocked cells primed by a mild heat shock. We observed an increased survival during severe heat stress, accompanied by a strong reduction of heat-induced cellular protein aggregates in cells lacking the ClpXP protease. We could demonstrate that the transcription factor Spx, a regulatory substrate of ClpXP, is critical for the prevention of protein aggregate formation because its regulon encodes redox chaperones, such as thioredoxin, required for protection against thiol-specific oxidative stress.

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.

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.

CtsR inactivation during thiol-specific stress in low GC, Gram+ bacteria.

CtsR, the global heat shock repressor in low GC, Gram+ bacteria, regulates a crucial subset of genes involved in protein quality control. CtsR de-repression occurs not only during heat stress but also during a variety of other environmental stresses, most notably thiol-specific oxidative stress. Here we report that McsA acts as a molecular redox switch that regulates CtsR de-repression via the activation of McsB.

Regulation of CtsR activity in low GC, Gram+ bacteria.

CtsR is the global transcriptional regulator of the core protein quality networks in low GC, Gram+ bacteria. Balancing these networks during environmental stress is of considerable importance for moderate survival of the bacteria, and also for virulence of pathogenic species. Therefore, inactivation of the CtsR repressor is one of the major cellular responses for fast and efficient adaptation to different protein stress conditions.

CtsR, the Gram-positive master regulator of protein quality control, feels the heat.

Protein quality networks are required for the maintenance of proper protein homeostasis and essential for viability and growth of all living organisms. Hence, regulation and coordination of these networks are critical for survival during stress as well as for virulence of pathogenic species. In low GC, Gram-positive bacteria central protein quality networks are under the control of the global repressor CtsR.

The number of G residues in the Bacillus subtilis pyrG initially transcribed region governs reiterative transcription-mediated regulation.

Derepression of pyrG expression in Bacillus subtilis involves CTP-sensitive reiterative transcription, which introduces up to 11 extra G residues at the 5' ends of pyrG transcripts. Insertion of three or more additional Gs at the 5' end of the pyrG initially transcribed region abolished reiterative transcription and caused constitutive expression.