Shunji Nakano

Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis

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.

A regulatory protein that interferes with activator-stimulated transcription in bacteria

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.

Redox-sensitive transcriptional control by a thiol/disulphide switch in the global regulator, Spx

The Spx protein is indispensable for survival of Bacillus subtilis under disulphide stress. Its interaction with the α‐subunit of RNA polymerase is required for transcriptional induction of genes that function in thiol homeostasis, such as thioredoxin (trxA) and thioredoxin reductase (trxB). The N‐terminal end of Spx contains a Cys–X–X–Cys (CXXC) motif, which is a likely target for redox‐sensitive control. We show here that Spx directly activates trxA and ‐B transcription by interacting with the RNA polymerase α‐subunit, but it does so only under an oxidized condition. The transcriptional activation by Spx requires formation of an intramolecular disulphide bond between two cysteine residues that reside in the CXXC motif. The mechanism of Spx‐dependent transcriptional activation is unique in that it does not involve initial Spx–DNA interaction.

SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis elegans.

The mitogen‐activated protein kinase (MAPK) pathway is a highly conserved signaling cascade that converts extracellular signals into various outputs. In Caenorhabditis elegans, asymmetric expression of the candidate odorant receptor STR‐2 in either the left or the right of two bilaterally symmetrical olfactory AWC neurons is regulated by axon contact and Ca2+ signaling. We show that the MAPK kinase (MAPKK) SEK‐1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK‐1 functions in a pathway downstream of UNC‐43 and NSY‐1, Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and MAPK kinase kinase (MAPKKK), respectively. Thus, the NSY‐1–SEK‐1–MAPK cascade is activated by Ca2+ signaling through CaMKII and establishes asymmetric cell fate decision during neuronal development.

Multiple Pathways of Spx (YjbD) Proteolysis in Bacillus subtilis

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 of Bacillus 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. The spx (formally yjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred by clpP and clpX. The level of Spx in wild-type cells grown in competence medium is low, and that in clpP mutants 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 the clpP mutation. 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 in clpX mutant to the same level as that observed in the clpP mutant. In contrast, a very low concentration of Spx was detected in a clpC mutant. 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 of spx, as measured by expression of spx-lacZ, was slightly increased by the clpX mutation. To exclude a possible effect of clpX and clpP on spx transcription, the spx gene was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter. 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.

Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans.

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system.

The Nitric Oxide-Responsive Regulator NsrR Controls ResDE-Dependent Gene Expression

The ResD-ResE signal transduction system is essential for aerobic and anaerobic respiration in Bacillus subtilis. ResDE-dependent gene expression is induced by oxygen limitation, but full induction under anaerobic conditions requires nitrite or nitric oxide (NO). Here we report that NsrR (formerly YhdE) is responsible for the NO-dependent up-regulation of the ResDE regulon. The null mutation of nsrR led to aerobic derepression of hmp (flavohemoglobin gene) partly in a ResDE-independent manner. In addition to its negative role in aerobic hmp expression, NsrR plays an important role under anaerobic conditions for regulation of ResDE-controlled genes, including hmp. ResDE-dependent gene expression was increased by the nsrR mutation in the absence of NO, but the expression was decreased by the mutation when NO was present. Consequently, B. subtilis cells lacking NsrR no longer sense and respond to NO (and nitrite) to up-regulate the ResDE regulon. Exposure to NO did not significantly change the cellular concentration of NsrR, suggesting that NO likely modulates the activity of NsrR. NsrR is similar to the recently described nitrite- or NO-sensitive transcription repressors present in various bacteria. NsrR likely has an Fe-S cluster, and interaction of NO with the Fe-S center is proposed to modulate NsrR activity.

The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ

Assembly of the tubulin‐like cytoskeletal protein FtsZ into a ring structure establishes the location of the nascent division site in prokaryotes. Factors that modulate FtsZ assembly are essential for ensuring the precise spatial and temporal regulation of cytokinesis. We have identified ClpX, the substrate recognition subunit of the ClpXP protease, as an inhibitor of FtsZ assembly in Bacillus subtilis. Genetic data indicate that ClpX but not ClpP inhibits FtsZ‐ring formation in vivo. In vitro, ClpX inhibits FtsZ assembly in a ClpP‐independent manner through a mechanism that does not require ATP hydrolysis. Together our data support a model in which ClpX helps maintain the cytoplasmic pool of unassembled FtsZ that is required for the dynamic nature of the cytokinetic ring. ClpX is conserved throughout bacteria and has been shown to interact directly with FtsZ in Escherichia coli. Thus, we speculate that ClpX functions as a general regulator of FtsZ assembly and cell division in a wide variety of bacteria.

Spx (YjbD), a negative effector of competence in Bacillus subtilis, enhances ClpC–MecA–ComK interaction

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.

Mutational Analysis of the Bacillus subtilis RNA Polymerase α C-Terminal Domain Supports the Interference Model of Spx-Dependent Repression

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-alphaCTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the alpha1 region of alphaCTD. Alanine-scanning mutagenesis of B. subtilis alphaCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-alphaCTD interaction and that residues Y263, C265, and K267 of the alpha1 region lie within overlapping ComA- and Spx-binding sites for alphaCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.