Peter Zuber

The Spx protein of Bacillus subtilis represses activator-stimulated transcription by interacting with the C-terminal domain of RNA polymerase (RNAP) α subunit. Its concentration increases in cells lacking the ATP-dependent protease, ClpXP, resulting in severe effects on growth and developmental processes. Microarray analysis was undertaken to identify genes that are induced or repressed when Spx interacts with RNAP. The induced genes included those encoding products known to function in maintaining thiol homeostasis. Two genes, thioredoxin ( trxA ) and thioredoxin reductase ( trxB ), are transcriptionally induced under conditions of thiol-specific oxidative (disulfide) stress by a mechanism involving Spx-RNAP interaction. Disulfide stress also results in an increase in Spx-dependent transcriptional repression. The increase in Spx activity in cells encountering disulfide stress is due in part to a posttranscriptional mechanism of spx control resulting in an increase in Spx concentration. An spx null mutant and a strain bearing an allele of rpoA that prevents Spx-RNAP interaction show hypersensitivity to disulfide stress. From these results, it is proposed that Spx is an activator that mobilizes the operations necessary to reverse the effects of oxidative damage, but it also serves as a negative regulator that causes the postponement of developmental programs and energy-consuming growth-related functions while the cell copes with the period of stress.

Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the α-subunit of RNA polymerase (RNAP). Here we report the discovery of an “anti-α” factor Spx in Bacillus subtilis that blocks transcriptional activation by binding to the α-C-terminal domain, thereby interfering with the capacity of RNAP to respond to certain activator proteins. Spx disrupts complex formation between the activator proteins ResD and ComA and promoter-bound RNAP, and it does so by direct interaction with the α-subunit. ResD- and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATP-dependent protease ClpXP. Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with α.

ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways. Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development ofBacillus subtilis. Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation. Thespx(formallyyjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred byclpPandclpX. The level of Spx in wild-type cells grown in competence medium is low, and that inclpPmutants is high. This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from theclpPmutation. In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx. Western blot analysis showed that Spx accumulated inclpXmutant to the same level as that observed in theclpPmutant. In contrast, a very low concentration of Spx was detected in aclpCmutant. An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH. However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx. Transcription ofspx, as measured by expression ofspx-lacZ, was slightly increased by theclpXmutation. To exclude a possible effect ofclpXandclpPonspxtranscription, thespxgene was placed under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible Pspacpromoter. In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx. Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP. The putative proteolysis by ClpXP might require another adapter protein. Spx probably is degraded by ClpCP under as yet unidentified conditions. This study suggests that the level of Spx is tightly controlled by two different ClpP proteases.

Summary: ComK, a key transcriptional regulator in the development of competence in Bacillus subtilis, is required for its own transcription as well as that of the late competence genes encoding proteins involved in DNA uptake. ComK is sequestered in a complex with ClpC and MecA until a peptide, ComS, accumulates in cells. ComS releases ComK from the inhibitory complex, thus allowing ComK to carry out its function as a transcriptional activator. Spx (formerly YjbD), a negative effector of competence, accumulates in clpP mutants. High concentrations of Spx may be responsible for the inability of clpP mutants to become competent because spx mutations are able to restore competence in the clpP mutant. In this paper, we showed, based on in vitro experiments, that Spx forms a quaternary complex with ClpC, MecA and ComK and enhances ComK binding to ClpC–MecA. Two ComS alanine substitution mutants (I13A and W43A), previously shown to be defective in vivo, were less efficient in releasing ComK from ClpC–MecA. The I13A mutant with a weaker binding affinity to MecA was inefficient in releasing ComK regardless of whether Spx was present. In contrast, the defect of the W43A mutant in dissociating ComK was more readily observed in the presence of Spx. Spx is a highly conserved protein among Gram‐positive bacteria, in which it may function closely with the protease adaptor protein, MecA.

Our introduction to prokaryotic gene expression has always focused on the operon and regulatory mechanisms that operate within enteric bacteria such as Escherichia coli, Salmonella species, and their phages. While operon organization and many of the components of regulatory networks are conserved in Gram-positive species, there exists unique features that set these organisms apart from the enterics. Two examples are presented herein: carbon catabolite control and regulation of RNA polymerase sigma subunit activity, are presented. The accompanying reviews highlight the diversity and novel aspects of genetic control in Gram-positive bacteria, with descriptions of quorum-sensing systems, transcriptional control, and RNA processing mechanisms.

Mutations in clpP and clpX have pleiotropic effects on growth and developmentally regulated gene expression in Bacillus subtilis. ClpP and ClpX are needed for expression of comK, encoding the competence transcription factor required for the expression of genes within the competence regulon. ClpP, in combination with the ATPase ClpC, degrades the inhibitor of ComK, MecA. Proteolysis of MecA is stimulated by a small protein, ComS, which interacts with MecA. Suppressor mutations (cxs) were isolated that bypass the requirement for clpX for comK expression. These were found also to overcome the defect in comK expression conferred by a clpP mutation. These mutations were identified as missense mutations (cxs‐5, ‐7 and ‐12) and a nonsense (UAG) codon substitution (cxs‐10) in the yjbD coding sequence in a locus linked to mecA. That a yjbD disruption confers the cxs phenotype, together with its complementation by an ectopically expressed copy of yjbD, indicated that the suppressor alleles bear recessive, loss‐of‐function mutations of yjbD. ClpP‐ and ClpX‐independent comK expression rendered by inactivation of yjbD was still medium‐dependent and required ComS. MecA levels in a clpP‐yjbD mutant were lower that those of clpP mutant cells and ComK protein concentration in the clpP mutant was restored to wild‐type levels by the yjbD mutation. Consequently, the yjbD mutation bypasses the defect in competence development conferred by clpP and clpX. YjbD protein is barely detectable in wild‐type cells, but is present in large amounts in the clpP mutant cells. The results suggest that the role of ClpP in competence development is to degrade YjbD protein so that ComS can productively interact with the MecA–ClpC–ComK complex. Alternatively, the result could suggest that YjbD has a negative effect on regulated proteolysis and that MecA is degraded independently of ClpP when YjbD is absent.

In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor σ^H form of RNA polymerase (RNAP). The induction of σ^H-dependent gene transcription requires the regulatory ATPase, ClpX. The ClpX-dependent post-exponential increase in σ^H activity is not dependent on the activator of sporulation gene expression, Spo0A. By determining the level of σ^H and σ^A in whole-cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in σ^A-RNAP holoenzyme. This is probably an indirect consequence of ClpX activity, because the ClpX-dependent decrease in σ^A-RNAP concentration does not occur in a spo0A abrB mutant. The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but σ^H-RNAP was observed to be more sensitive to ClpX-dependent stimulation than σ^A-RNAP. No difference in transcriptional activity was observed in single-cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed- and open-promoter complex formation. ClpX is proposed to function indirectly in the displacement of σ^A from core RNAP and to act directly in the stimulation of σ^H-dependent transcription in sporulating B. subtilis cells.

The Bacillus subtilis 168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against Listeria monocytogenes . Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene sboA and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process. The genes of the sbo-alb operon are believed to function in the synthesis and maturation of subtilosin. Nonpolar mutations introduced into each of the alb genes resulted in loss or reduction of subtilosin production. sboA , albA , and albF mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin. Mutations in albB , - C , and - D resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions. A new gene, sboX , encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of sboA . Construction of an sboX-lacZ translational fusion and analysis of its expression indicate that sboX is induced in stationary phase of anaerobic cultures of JH642. An in-frame deletion of the sboX coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin. The results of this investigation show that the sbo-alb genes are required for the mechanisms of subtilosin synthesis and immunity.