Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the ␣-subunit of RNA polymerase (RNAP). Here we report the discovery of an ''anti-␣'' factor Spx in Bacillus subtilis that blocks transcriptional activation by binding to the ␣-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 ␣-subunit. ResD-and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATPdependent protease ClpXP. Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with ␣.Spx ͉ RNA polymerase ͉ ␣-subunit ͉ Bacillus subtilis ͉ transcriptional activation T ranscriptional activation in bacteria involves contacts between DNA-bound activators and promoter-bound RNA polymerase (RNAP). Most such interactions require the ␣-subunit of RNAP, which possesses several activator-interaction surfaces within its C-terminal domain (CTD) (1, 2). One such activator is ComA of the bacterium Bacillus subtilis (3-5), a response regulator, required for transcription of genes involved in the development of genetic competence (6). In response to high cell density, ComA becomes phosphorylated by interaction with its cognate histidine kinase, ComP, that is activated when it binds the pheromone ComX (7-9). ComA then activates the transcription initiation of the srf operon, which encodes the competence regulatory peptide ComS (10, 11). ComS serves to release the transcriptional activator ComK from its inhibitory complex composed of the proteins MecA and ClpCP (10-13), so that ComK can stimulate transcription of genes required for DNA uptake in competent cells (14). Interestingly, ComAdependent transcription of srf requires the ATP-dependent protease ClpXP (15, 16), which functions to eliminate the 15.4-kDa Spx protein (refs. 17 and 18; see Results). A mutation in clpX blocks ComA-activated transcription and has severe effects on growth and development (17). These pleiotropic effects of ClpXP absence can be suppressed either by the elimination of Spx or by missense mutations in the rpoA gene that encodes the RNAP ␣-subunit (refs. 16 and 17; see Results). This latter finding suggested that Spx exerts its negative effect on ComA-mediated transcription, and other transcriptional activation systems, by interaction with RNAP. A similar relationship between Spx and the ␣-subunit of RNAP was observed for ResD-activated transcription (see Results). ResD, like ComA, is a response regulator and transcriptional activator. It is part of the ResDE two-component signal transduction system that is required for the transcription of genes that are induced in response to oxygen limitation (19).In this article we show that Spx interferes with activatorstimulated transcription by interaction with the RNAP ␣-CTD, a mechanism of transcriptional repression ...

SummaryRecently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/ glyoxalases, oxidoreductases and the azoreductase encoding yvaB (azoR2) gene in response to thiolspecific stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ (azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho-and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles.

SummaryCatechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the DykvE mutant revealed the upregulation of ykcA (mhqA), ydfNOP (mhqNOP), yodED (mhqED) and yvaB (azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified s A -type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter-bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA-protein binding complexes. DNAbinding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the DykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis.

The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P 3 promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P 3 promoter. Expression of an spx-bgaB fusion carrying the P 3 promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P 3 transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P 3 promoter DNA was used as a template. Point mutations at positions within the P 3 promoter relieved YodB-dependent repression, while a point mutation at position ؉24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P 3 ؊10 and ؊35 regions, while PerR binds to a region downstream of the P 3 transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.Spx is a highly conserved transcriptional regulatory protein of low-GC-content gram-positive bacteria (2,4,7,29,36,40). It does not possess sequence-specific DNA-binding activity but instead directly targets RNA polymerase (RNAP) (26,28), an interaction that interferes with contact between transcriptional activators (26, 39) and RNAP while activating transcription at promoters of genes whose products function in intracellular thiol homeostasis (24,25) and responses to encounters with toxic oxidants (25,29,33,34). Recent studies suggested that Spx plays a role in cellular invasion by a microbial pathogen (2). Spx does not belong to any other known family of transcriptional regulatory proteins but resembles the arsenate reductase ArsC of plasmid R773 in terms of its primary and higher-order structure (18,28,40).The spx gene resides in the yjbC-spx operon of the Bacillus subtilis genome and is transcribed from at least five promoters by four forms of RNAP holoenzyme ( A , B , M , and W ). Induction of spx expression has been associated with phosphate starvation, ethanol, and oxidative stress (1, 25, 35), while induction of the spx regulon is activated by a variety of stress conditions that include heat shock, salt stress, oxidative stress, and toxic phe...

A genetically related pair of human head and neck cancer (HNSCC) cell lines derived from the same patient at different stages of disease was used to investigate the role of extracellular matrix, integrin, and CXCL12-CXCR4 receptor interactions and their signal pathways in MMP-2 and MMP-9 activation and cell invasion. We found that collagen I enhanced MMP-2 and MMP-9 secretion in both primary and metastatic HNSCC cells. Collagen I acted through α(2)β(1) integrin to activate tyrosine kinases, protein kinase C, ERK1/2, and p38, which in turn activated MMP-2 and MMP-9 production. The signaling function was also involved in the enhancement of cell invasion. Experiments using cocultures between live and fixed cells demonstrated that direct contact between tumor and fibroblast cells was required to activate MMP-2 and MMP-9 secretion in both tumor cells and fibroblasts. The augmentation appears specific for MMP-2. Fibroblasts seem to be responsible for the increased MMP-2 in the coculture. In addition, fibroblast or tumor cell-conditioned media upregulated the secretion of MMP-2 and MMP-9 in HNSCC cells. These findings indicate that autocrine and paracrine factors are involved in the augmented secretion of MMPs in coculture. We also found that CXCL12-enhanced HNSCC cell invasion through paracrine-activated CXCR4, which triggered MMP-dependent cell invasion. Together, our results suggest that cell-matrix and cell-cell interactions including autocrine and paracrine factors play important roles in the invasive behavior of HNSCC via upregulation of MMP-2 and MMP-9.

Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis. Its target is RNA polymerase, where it contacts the ␣ subunit C-terminal domain. Recently, evidence was presented that Spx participates in sulfate-dependent control of organosulfur utilization operons, including the ytmI, yxeI, ssu, and yrrT operons. The yrrT operon includes the genes that function in cysteine synthesis from S-adenosylmethionine through intermediates S-adenosylhomocysteine, ribosylhomocysteine, homocysteine, and cystathionine. These operons are also negatively controlled by CymR, the repressor of cysteine biosynthesis operons. All of the operons are repressed in media containing cysteine or sulfate but are derepressed in medium containing the alternative sulfur source, methionine. Spx was found to negatively control the expression of these operons in sulfate medium, in part, by stimulating the expression of the cymR gene. In addition, microarray analysis, monitoring of yrrT-lacZ fusion expression, and in vitro transcription studies indicate that Spx directly activates yrrT operon expression during growth in medium containing methionine as sole sulfur source. These experiments have uncovered additional roles for Spx in the control of gene expression during unperturbed, steadystate growth.The global regulator Spx of Bacillus subtilis functions in the oxidative stress response by activating transcription of genes that function in thiol homeostasis (33,34,49). In this capacity, it is required for the transcriptional activation of the thioredoxin (trxA) and thioredoxin reductase (trxB) genes in response to disulfide stress. It also represses genes that function in a variety of metabolic and developmental pathways (34,35). The activity and synthesis of Spx are stimulated when cells undergo accelerated disulfide generation resulting from encounters with toxic oxidants. The accumulated Spx interacts with RNA polymerase (RNAP) in part by contacting residues of the alpha subunit C-terminal domain (CTD), while showing no sequence-specific DNA-binding activity (36). An important feature of Spx is the N-terminal CxxC redox disulfide motif that controls Spx activity. Transcriptional activation from the trxA and trxB promoters requires that the two cysteines be in the oxidized, disulfide state. The presence of a reductant that converts the cysteines to the thiol state results in loss of transcription-stimulating activity (33). It is currently not known how the interaction of oxidized Spx protein with RNA polymerase activates transcription at promoters under Spx positive control.In addition to its role in oxidative stress, recent studies indicate that Spx functions as a regulator of gene expression in cells undergoing steady-state growth. Spx was found to negatively affect the transcription of genes that function in the utilization of organosulfur compounds as alternative sources of sulfur (13). Such operons are repressed in minimal glucose media containing either of the two preferred sulfur sources, sulfate and cys...

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