Crystal structure of the<i>Bacillus subtilis</i>anti-alpha, global transcriptional regulator, Spx, in complex with the α C-terminal domain of RNA polymerase

Newberry, Kate J.; Nakano, Shunji; Zuber, Peter; Brennan, Richard G.

doi:10.1073/pnas.0506592102

Cited by 81 publications

(135 citation statements)

References 41 publications

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“…Spx controls gene expression by interacting directly with the ␣ subunit of the RNA polymerase (45). The Spx-controlled trfA gene was recently shown to be required for the ability of subinhibitory concentrations of ␤-lactam antibiotics to induce the hVISA phenotype, raising the intriguing possibility that the elevated Spx level may contribute to the decreased susceptibility to daptomycin, vancomycin, and ␤-lactams (46,47).…”

Section: Discussionmentioning

confidence: 99%

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Bæk

Thøgersen

Mogenssen

et al. 2015

Antimicrob Agents Chemother

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dDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A 477 D) that decreased susceptibility not only to daptomycin but also to vancomycin, ␤-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S 295 L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits. Staphylococcus aureus is a commensal bacterium that can cause a variety of both localized and more invasive infections. Due to its profound ability to acquire resistance to clinically relevant antibiotics, S. aureus remains a major clinical challenge worldwide (1). The most challenging antimicrobial resistance issue in S. aureus has been the evolution and dissemination of methicillinresistant S. aureus (MRSA) strains first in hospitals and later in the general community (1). MRSA infections are typically treated with last-line antibiotics, such as vancomycin and, more recently, daptomycin (DAP), a cyclic lipopeptide derived from Streptomyces roseosporus with bactericidal activity against a broad range of Gram-positive bacteria (2, 3). The mechanism of action of daptomycin has not been fully elucidated, but it is generally accepted that daptomycin inserts into the cytoplasmic membrane of Grampositive bacteria via a calcium-dependent pathway, leading to potassium efflux, membrane depolarization, and, eventually, cell death (2).The development of daptomycin nonsusceptibility (DAP-NS) in S. aureus seems to be a relatively rare phenomenon not involving horizontal gene transfer but, instead, taking place by a stepwise acqu...

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Section: Discussionmentioning

confidence: 99%

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Bæk

Thøgersen

Mogenssen

et al. 2015

Antimicrob Agents Chemother

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“…One codon change conferring the clpX suppressor phenotype was Y263C, in the ␣1 helix of the ␣CTD. The other clpX suppressor locus is the spx gene (16), the product of which interacts with the RNAP to affect transcription initiation (25,27). Subsequent structural analysis confirmed that Spx interacts with the ␣CTD of RNAP and that the binding surface includes residue Y263 of the ␣ subunit (27).…”

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Mutational Analysis of theBacillus subtilisRNA Polymerase α C-Terminal Domain Supports the Interference Model of Spx-Dependent Repression

et al. 2006

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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 (␣CTD). 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-␣CTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the ␣1 region of ␣CTD. Alanine-scanning mutagenesis of B. subtilis ␣CTD 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-␣CTD interaction and that residues Y263, C265, and K267 of the ␣1 region lie within overlapping ComA-and Spx-binding sites for ␣CTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.The spx gene of Bacillus subtilis was identified as the site of mutations that overcome the requirement for the protease ClpXP in the expression of genes that are transcriptionally activated by response regulator proteins (16,25). The srf operon of B. subtilis, which contains genes encoding products that function in the control of competence development and in nonribosomal peptide synthesis (4,11,17,33), is activated by the ComPA two-component signal transduction system (5, 6). ComA is a response regulator that becomes phosphorylated by interaction with the histidine kinase ComP when the latter autophosphorylates in response to the peptide pheromone ComX (9). This quorum-sensing system converts ComA to active ComA phosphate (ComAϳP), which interacts as two dimers with the two ComA box elements residing upstream of the srf operon promoter (21, 30). ComA-dependent transcriptional activation is one of the regulatory events in B. subtilis that are negatively affected by Spx (25).Competence development, as well as several other transition state processes of B. subtilis, is severely impaired in strains bearing mutations in clpX or clpP (12, 15, 16). Likewise, srf operon expression is diminished in clpX and clpP mutant cells (16,19). Some of the suppressor mutations resulting in restored srf expression in a clpX background mapped to the rpoA gene, which encodes the RNAP ␣ subunit. Codon substitutions in the region encoding the C-terminal domain of ␣ (␣CTD) were uncovered through this suppressor analysis (19). One codon change conferring the clpX suppressor phenotype was Y263C, in the ␣1 helix of the ␣CTD. The other clpX suppressor locus is the spx gene (16), the product of which interacts with the RNAP to affect transcription initiation (25,27). Subsequent structural analysis confirmed that Spx interacts with the ␣CTD of RNAP and that the binding surface includes residue Y263 of the ␣ subunit (27). T...

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“…This leads to induction of the Spx regulon, which includes trxA (encoding thioredoxin), trxB (thioredoxin reductase), and other genes that function in the oxidative stress response and in cysteine biosynthesis (10, 34). Spx-dependent activation of trxA and trxB requires interaction of the protein with RNA polymerase (RNAP) holoenzyme (33,37). This interaction also results in repression of operons that require an activator for transcription initiation (35, 54).…”

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confidence: 99%

The YjbH Protein ofBacillus subtilisEnhances ClpXP-Catalyzed Proteolysis of Spx

et al. 2009

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The global transcriptional regulator Spx of Bacillus subtilis is controlled at several levels of the gene expression process. It is maintained at low concentrations during unperturbed growth by the ATP-dependent protease ClpXP. Under disulfide stress, Spx concentration increases due in part to a reduction in ClpXPcatalyzed proteolysis. Recent studies of Larsson and coworkers (Mol. Microbiol. 66:669-684, 2007) implicated the product of the yjbH gene as being necessary for the proteolytic control of Spx. In the present study, yeast two-hybrid analysis and protein-protein cross-linking showed that Spx interacts with YjbH. YjbH protein was shown to enhance the proteolysis of Spx in reaction mixtures containing ClpXP protease but not ClpCP protease. An N-terminal truncated form of YjbH with a deletion of residues 1 to 24 (YjbH ⌬1-24 ) showed no proteolysis enhancement activity. YjbH is specific for Spx as it did not accelerate proteolysis of the ClpXP substrate green fluorescent protein (GFP)-SsrA, a GFP derivative with a C-terminal SsrA tag that is recognized by ClpXP. Using inductively coupled plasma atomic emission spectroscopy and 4-(2-pyridylazo) resorcinol release experiments, YjbH was found to contain zinc atoms. Zinc analysis of YjbH ⌬1-24 revealed that the N-terminal histidine-rich region is indispensable for the coordination of at least one Zn atom. A Zn atom coordinated by the N-terminal region was rapidly released from the protein upon treatment with a strong oxidant. In conclusion, YjbH is proposed to be an adaptor for ClpXP-catalyzed Spx degradation, and a model of YjbH redox control involving Zn dissociation is presented.In the spore-forming bacterium Bacillus subtilis, the protein Spx is a global transcriptional regulator that exerts both positive and negative control of multiple genes during thiol-specific oxidative stress (34, 57). Cells undergoing disulfide stress exhibit an elevated Spx concentration and heightened Spx activity. This leads to induction of the Spx regulon, which includes trxA (encoding thioredoxin), trxB (thioredoxin reductase), and other genes that function in the oxidative stress response and in cysteine biosynthesis (10, 34). Spx-dependent activation of trxA and trxB requires interaction of the protein with RNA polymerase (RNAP) holoenzyme (33,37). This interaction also results in repression of operons that require an activator for transcription initiation (35, 54).The spx gene was first identified as a suppressor locus of clpP and clpX mutations (32). ClpX proteins form hexameric, ringshaped complexes and belong to the AAAϩ (for ATPases associated with a variety of cellular activities) Clp/Hsp100 family of proteins (2). ClpX is the ATPase and substrate-binding component of the ClpXP protease. ClpX also functions as an unfoldase and translocase that can denature and deliver substrate proteins to the proteolytic chamber, which is composed of two heptameric rings of the ClpP subunits (45). In wild-type cells under normal growth conditions, Spx is present at nearly undetectable l...

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Crystal structure of theBacillus subtilisanti-alpha, global transcriptional regulator, Spx, in complex with the α C-terminal domain of RNA polymerase

Cited by 81 publications

References 41 publications

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Mutational Analysis of theBacillus subtilisRNA Polymerase α C-Terminal Domain Supports the Interference Model of Spx-Dependent Repression

The YjbH Protein ofBacillus subtilisEnhances ClpXP-Catalyzed Proteolysis of Spx

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