The Global Regulator Spx Functions in the Control of Organosulfur Metabolism in<i>Bacillus subtilis</i>

Choi, Soon Yong; Reyes, Dindo; Leelakriangsak, Montira; Zuber, Peter

doi:10.1128/jb.00443-06

Cited by 38 publications

(40 citation statements)

References 50 publications

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“…For overproduction and purification of Spx proteins in E. coli, ER2566 strains (New England Biolabs) bearing spx-overexpressing plasmids were grown at 37°C in LB liquid medium. Antibiotic concentrations used were as previously reported (6).…”

Section: Methodsmentioning

confidence: 99%

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Lin

Zuber

2012

J Bacteriol

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Spx activates transcription initiation inT hroughout phylogeny, positive transcriptional control plays a important role in cellular decision-making (45). The mechanisms that activate transcription initiation in response to environmental and metabolic signal inputs are varied and involve complex sensory functions linked to specific macromolecular interactions conducted at targeted chromosomal loci. For many regulatory systems, these interactions involve contacts between transcriptional regulators and RNA polymerase (RNAP). In prokaryotes, RNA polymerase, composed of ␤, ␤=, , 2 ␣, and subunits, contains multiple target surfaces that can engage positive regulatory proteins, which function to direct RNAP to the specific regulatory regions of genes that are under their control (17). The ␣ subunit, bearing two domains that occupy the protein's N and C termini (NTD and CTD, respectively), is a common target for regulatory protein interaction (18). A classic example is the class I and II positive control exerted by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP), which interacts with a specific cis-acting element upstream of promoter DNA as a dimeric complex that recruits RNAP by direct interaction with RNAP ␣CTD (4).While direct interaction between DNA-bound regulators and RNAP is a common mechanism of positive transcriptional control, mechanisms of RNAP appropriation and "prerecruitment" have been reported that involve initial RNAP-regulator interaction prior to promoter engagement (2). Proteins encoded in bacteriophage genomes, such as NS4SSB of phage N4 (26) and AsiA of phage T4 (15), target RNAP without contacting DNA and yet participate in gene-specific transcriptional activation. The SoxS protein that mediates the bacterial response to superoxide, and the MarA protein that functions in control of multidrug resistance mechanisms, first contacts the RNAP ␣ subunit prior to sequencespecific DNA interaction (11,25). Spx, a protein of low-GC-content Gram-positive bacteria that serves as a transcriptional activator of genes that function in thiol-specific oxidative stress (11,25,51), also exerts control of RNAP through a prerecruitment mechanism (31).Spx governs a large regulon of genes that includes those encoding thioredoxin (trxA), thioredoxin reductase (trxB) (34), and methionine sulfoxide reductase (49) and genes whose products function in bacillithiol biosynthesis (9), cysteine biosynthesis (35), and iron uptake and metabolism (52). Spx has also been linked to control of biofilm-and virulence-related functions (19,39). Spx is under multiple levels of control that operate at the transcriptional and posttranscriptional level. Multiple forms of RNAP target the operon in which the spx gene resides (8,41). The spx gene itself is under negative transcriptional control involving two repressors, YodB and PerR, that control the transcriptional response to toxic electrophiles and peroxide, respectively (23). Spx protein is under tight proteolytic control involving the ATP-dependent protease ClpXP (35, 36) an...

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Section: Methodsmentioning

confidence: 99%

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Lin

Zuber

2012

J Bacteriol

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“…For overproduction and purification of Spx proteins in E. coli strain ER2566, cells were grown at 37°C in LB liquid medium. Antibiotic concentrations used were as previously reported (35,36). For trpC2 pheA1 phenotype confirmation, JH642 derivatives were streaked on a TSS minimal medium agar plate with or without tryptophan and phenylalanine supplements.…”

Section: Methodsmentioning

confidence: 99%

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Lin

Walthers

Zuber

2013

Journal of Bacteriology

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Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP ␣ subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at ؊44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP A subunit from a ؊35-like element upstream of the trxB (thioredoxin reductase) promoter to positions ؊36 and ؊11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP. Bacterial responses to environmental and metabolic changes often involve gene regulation at the level of transcription initiation. The essential enzyme catalyzing this first step of gene expression, RNA polymerase (RNAP), is targeted by a variety of regulatory factors that direct RNAP activity to specific transcription units (1). The core RNAP, bearing the catalytic component, is composed of large subunits, ␤ and ␤=, the ␣ subunit dimer, and the subunit. Interaction of core RNAP with the subunit gives rise to the holoenzyme endowed with gene promoter specificity (2, 3). Regulatory factors that are controlled by signal sensing/ transducing systems that mediate stress responses can target one or more of the RNAP subunits to modulate activity and promoter specificity. Some regulatory factors exert positive control by engaging promoter DNA and recruiting RNAP through direct protein-RNAP subunit interaction (1). There is a growing list of regulatory proteins that prerecruit or appropriate RNAP by first contacting holoenzyme before mediating DNA target recognition (4). One of them, AsiA of phage T4, serves to mediate contact between RNAP subunit and a phage-encoded DNA-bound factor, MotA, which triggers prereplicative phage gene transcription (5). The DksA/Gre family proteins target the RNAP active site to affect transcription initiation and elongation at specific promoters (6). SoxS and related proteins engage in prerecruitment by interacting with holoenzyme before contacting specific cis-acting promoter elements (7). Other factors, such as Crl and 6S RNA, stabilize or inhibit specific holoenzyme forms beari...

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“…Plasmids pSN16 (spx wild-type promoter) (27), pML42 [spx(TϪ19G)], pML44 [spx(TϪ26A)], and pML48 [spx(Tϩ24C)] (15a) were used as PCR templates. The DNA probe for yrrT was made as described previously (3). The coding-strand primer (oML02-25) (15a) was treated with T4 polynucleotide kinase and [␥- 32 P]ATP before the PCRs.…”

Section: Methodsmentioning

confidence: 99%

Dual Negative Control ofspxTranscription Initiation from the P₃Promoter by Repressors PerR and YodB inBacillus subtilis

2007

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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...

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The Global Regulator Spx Functions in the Control of Organosulfur Metabolism inBacillus subtilis

Cited by 38 publications

References 50 publications

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Dual Negative Control ofspxTranscription Initiation from the P₃Promoter by Repressors PerR and YodB inBacillus subtilis

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The Global Regulator Spx Functions in the Control of Organosulfur Metabolism inBacillus subtilis

Cited by 38 publications

References 50 publications

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

Dual Negative Control ofspxTranscription Initiation from the P3Promoter by Repressors PerR and YodB inBacillus subtilis

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Dual Negative Control ofspxTranscription Initiation from the P₃Promoter by Repressors PerR and YodB inBacillus subtilis