Alexander K. W. Elsholz

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.

The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

ABSTRACT 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. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. IMPORTANCE Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds. Staphylococcus aureus is a leading cause of multiantibiotic-resistant nosocomial infections and is often found growing as a biofilm in catheters and chronic wounds. Biofilm formation is an important pathogenicity strategy that enhances resistance to antimicrobials, thereby limiting treatment options and ultimately contributing to increased morbidity and mortality. Cells in a biofilm are held together by an extracellular matrix that consists in whole or in part of protein, but the nature of the proteins in the S. aureus matrix is not well understood. Here we postulate that S. aureus recycles proteins from the cytoplasm to form the extracellular matrix. This strategy, of cytoplasmic proteins moonlighting as matrix proteins, could allow enhanced flexibility and adaptability for S. aureus in forming biofilms under infection conditions and could promote the formation of mixed-species biofilms in chronic wounds.

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.

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. Here, we provide evidence that CtsR activity during heat stress is mediated by intrinsic heat sensing through a glycine‐rich loop, probably in all Gram‐positive species. Moreover, a function for the recently identified arginine kinase McsB is confirmed, however, not for initial inactivation and dissociation of CtsR from the DNA, but for heat‐dependent auto‐activation of McsB as an adaptor for ClpCP‐mediated degradation of CtsR.

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. In the absence of EPS, the kinase is inactivated by autophosphorylation. The presence of EPS inhibits autophosphorylation and instead promotes the phosphorylation of a glycosyltransferase in the biosynthetic pathway, thereby stimulating the production of EPS. Thus, EPS production is subject to a positive feedback loop that ties its synthesis to its own concentration. Tyrosine kinase-mediated self-regulation could be a widespread feature of the control of exopolysaccharide production in bacteria.

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. Once critical thiols of McsA become oxidized, the strong interaction between McsA and McsB is interrupted and free McsB is no longer inhibited by McsA, resulting in the inactivation of CtsR. This mechanism differs significantly from inactivation of CtsR during heat stress demonstrating a dual activity control of CtsR. Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation.

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. Consequently B. subtilis cells grown in the absence of oxygen were more protected against severe heat shock and much less protein aggregates were detected compared to aerobically grown cells. The presented results indicate that in B. subtilis Spx and its regulon plays not only an important role for oxidative but also for heat stress response and thermotolerance development. In addition, our experiments suggest that the protection of misfolded proteins from thiol oxidation during heat shock can be critical for the prevention of cellular protein aggregation in vivo.

Activity Control of the ClpC Adaptor McsB in Bacillus subtilis

Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.

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. Historically, CtsR inactivation was mainly studied for the heat stress response, and recently it has been shown that CtsR is an intrinsic thermosensor. Moreover, it has been demonstrated that CtsR degradation is regulated by a two-step mechanism during heat stress, dependent on the arginine kinase activity of McsB. Interestingly, CtsR is also inactivated during oxidative stress, but by a thiol-dependent regulatory pathway. These observations suggest that dual activity control of CtsR activity has developed during the course of evolution.

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. We isolated spontaneous mutants of B. subtilis that survive in the presence of a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNA(Tyr) charging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the tyrS riboswitch, as well as by a mutation that transformed a tRNA(Phe) into a tyrS riboswitch ligand. The most potent mutation, a substitution near the tyrosine recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNA(Tyr), thus facilitating the exclusion of D-tyrosine from protein biosynthesis in cells that lack D-aminoacyl-tRNA deacylase. IMPORTANCE Proteins are composed of L-amino acids. Mischarging of tRNAs with D-amino acids or the misincorporation of D-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to D-amino acids and their mechanisms of action.