Adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH‐enhanced proteolysis of the regulator Spx by ClpXP

The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP‐dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C‐terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C‐terminus showed that a 28‐residue C‐terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH‐targeted 28 residue segment of the Spx C‐terminus bears a ClpXP‐recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C‐terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild‐type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of helix α6, exposes the C‐terminus of Spx for recognition and proteolysis by ClpXP.     

YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO)

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.     

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus

Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress-associated sigma B factor (σ^B). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σ^B activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs.     

Desiccation tolerance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Staphylococcus aureus is a multidrug-resistant pathogen that not only causes a diverse array of human diseases, but also is able to survive in potentially dry and stressful environments, such as the human nose, on skin and on inanimate surfaces such as clothing and surfaces. This study investigated parameters governing desiccation tolerance of S. aureus and identified several components involved in the process. Initially, the role of environmental parameters such as temperature, growth phase, cell density, desiccation time and protectants in desiccation tolerance were determined. This established a robust model of desiccation tolerance in which S. aureus has the ability to survive on dry plastic surfaces for more than 1,097 days. Using a combination of a random screen and defined mutants, clpX, sigB and yjbH were identified as being required for desiccation tolerance. ClpX is a part of the ATP-dependent ClpXP protease, important for protein turnover, and YjbH has a proposed linked function. SigB is an accessory sigma factor with a role in generalized stress resistance. Understanding the molecular mechanisms that govern desiccation tolerance may determine the break points to be exploited to prevent the spread of this dangerous pathogen in hospitals and communities.     

Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus

The pathogenesis of staphylococcal infections is multifactorial. Golden pigment is an eponymous feature of the human pathogen Staphylococcus aureus that shields the microbe from oxidation-based clearance, an innate host immune response to infection. Here, we screened a collection of S. aureus transposon mutants for pigment production variants. A total of 15 previously unidentified genes were discovered. Notably, disrupting metabolic pathways such as the tricarboxylic acid cycle, purine biosynthesis, and oxidative phosphorylation yields mutants with enhanced pigmentation. The dramatic effect on pigment production seems to correlate with altered expression of virulence determinants. Microarray analysis further indicates that purine biosynthesis impacts the expression of ∼400 genes involved in a broad spectrum of functions including virulence. The purine biosynthesis mutant and oxidative phosphorylation mutant strains exhibit significantly attenuated virulence in a murine abscess model of infection. Inhibition of purine biosynthesis with a known small-molecule inhibitor results in altered virulence gene expression and virulence attenuation during infection. Taken together, these results suggest an intimate link between metabolic processes and virulence gene expression in S. aureus. This study also establishes the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival.Staphylococcus aureus causes a variety of infections in humans, ranging from minor skin and wound infections to life-threatening diseases (31). The pathogenesis of staphylococcal infections is a multifactorial process that depends on the expression of different virulence factors controlled by multiple regulatory systems in conjunction with environmental and nutritional signals (46). The high degree of variability in the expression of virulence genes is modulated by a complex network regulated by factors such as the agr locus (RNAIII), SarA, and SigB (5, 9), which allows the bacterium to adapt to changing environmental conditions for survival and developing infection.The species epithet of S. aureus reflects its characteristic surface pigmentation (aureus, meaning “golden” in Latin) (43). The yellowish-orange (golden) pigment produced by S. aureus has been linked to virulence, owing to its antioxidant property (29, 30). The golden pigmentation of S. aureus is the product of a C₃₀ triterpenoid carotenoid biosynthesis pathway, and the carotenoid pigment biosynthesis genes are organized in an operon crtOPQMN controlled by the alternative sigma factor SigB (3, 39). Since many virulence genes are coordinately regulated in S. aureus (5, 9, 31), we hypothesized that genes affecting pigmentation may also influence the production of virulence determinants and have an impact on the pathogenesis of S. aureus.Herein, we present an analysis of S. aureus golden pigment biosynthesis and regulatory pathways at the genomic level by screening the Phoenix (ΦNΞ) library, a collection of defined transposon insertions into 1,812 open reading frames of S. aureus strain Newman (1). This study indicates an intimate link between metabolic processes and virulence gene expression. It demonstrates the importance of purine biosynthesis and oxidative phosphorylation for in vivo survival and pathogenesis of S. aureus. Our results show that targeting purine biosynthesis is a promising strategy to develop anti-S. aureus therapies.     

The σB signalling activation pathway in the enteropathogen Clostridioides difficile

Clostridium difficile is the main cause of antibiotic-associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σ^B. Following stresses, σ^B activation requires a PP2C phosphatase to dephosphorylate the anti-anti-sigma factor RsbV that allows its interaction with the anti-sigma factor RsbW and the release of σ^B. In this work, we studied the signalling pathway responsible for the activation of σ^B in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σ^B. We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σ^B activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σ^B activity, depletion of the essential iron–sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σ^B activation. Finally, we observed that σ^B is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a ‘bet-hedging’ strategy allowing a better chance for the cells to survive adverse conditions.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.