Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes.

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 Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, 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 S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

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

Initiation 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.

Time-Resolved Proteome Analysis of Listeria monocytogenes 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 de novo synthesis in an intracellular human pathogen. We showcase this method by investigating the Listeria monocytogenes proteome in the presence and absence of the AAA+ chaperone protein ClpC. ClpC associates with the peptidase ClpP to form an ATP-dependent protease complex and has been shown to play a role in virulence development in L. monocytogenes. However, the mechanism by which ClpC is involved in the survival and proliferation of intracellular L. monocytogenes remains elusive. Employing this new method, we observed extensive proteome remodeling in L. monocytogenes upon interaction with the host, supporting the hypothesis that ClpC-dependent protein degradation is required to initiate bacterial adaptation mechanisms. We identified more than 100 putative ClpC target proteins through their stabilization in a clpC deletion strain. Beyond the identification of direct targets, we also observed indirect effects of the clpC deletion on the protein abundance in diverse cellular and metabolic pathways, such as iron acquisition and flagellar assembly. Overall, our data highlight the crucial role of ClpC for L. monocytogenes adaptation to the host environment through proteome remodeling. IMPORTANCE Survival and proliferation of pathogenic bacteria inside the host depend on their ability to adapt to the changing environment. Profiling the underlying changes on the bacterial proteome level during the infection process is important to gain a better understanding of the pathogenesis and the host-dependent adaptation processes. The cellular protein abundance is governed by the interplay between protein synthesis and decay. The direct readout of these events during infection can be accomplished using pulsed stable-isotope labeling by amino acids in cell culture (SILAC). Combining this approach with tandem-mass-tag (TMT) labeling enabled multiplexed and time-resolved bacterial proteome quantification during infection. Here, we applied this integrated approach to investigate protein turnover during the temporal progression of adaptation of the human pathogen L. monocytogenes to its host on a system-wide scale. Our experimental approach can easily be transferred to probe the proteome remodeling in other bacteria under a variety of perturbations.

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.

Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis.

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 Bacillus subtilis. 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. 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.

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. 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.

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.

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.

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.

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.

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.

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.

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.

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.