scholarly journals Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

2005 ◽  
Vol 187 (12) ◽  
pp. 4042-4049 ◽  
Author(s):  
Kyle N. Erwin ◽  
Shunji Nakano ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcriptional Control ◽  
Sulfur Metabolism ◽  
Growth Conditions ◽  
Sulfur Source ◽  
Dependent Manner ◽  
Α Subunit ◽  
A Genome

ABSTRACT Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions.

scholarly journals The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

scholarly journals Genome-Wide Analysis of the Stationary-Phase Sigma Factor (Sigma-H) Regulon of Bacillus subtilis

2002 ◽  
Vol 184 (17) ◽  
pp. 4881-4890 ◽  
Author(s):  
Robert A. Britton ◽  
Patrick Eichenberger ◽  
Jose Eduardo Gonzalez-Pastor ◽  
Paul Fawcett ◽  
Rita Monson ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Transcriptional Profiling ◽  
Open Reading Frames ◽  
Nutrient Sources ◽  
Genome Wide ◽  
A Genome ◽  
Sigma H

ABSTRACT Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

scholarly journals Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Cierra A. Birch ◽  
Madison J. Davis ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Stress Induction ◽  
Content Type ◽  
Terminal Domain ◽  
Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

scholarly journals Crystal structure of the in vivo-assembled Bacillus subtilis Spx/RNA polymerase α subunit C-terminal domain complex

2009 ◽  
Vol 168 (2) ◽  
pp. 352-356 ◽  
Author(s):  
Valerie Lamour ◽  
Lars F. Westblade ◽  
Elizabeth A. Campbell ◽  
Seth A. Darst
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Subunit C ◽  
Terminal Domain

scholarly journals Uncovering New Metabolic Capabilities of Bacillus subtilis Using Phenotype Profiling of Rifampin-Resistant rpoB Mutants

2007 ◽  
Vol 190 (3) ◽  
pp. 807-814 ◽  
Author(s):  
Amy E. Perkins ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Metabolic Profiling ◽  
Transcriptional Control ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Mutant Strains ◽  
Utilization Patterns ◽  
Flow Of Information

ABSTRACT RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase β subunit gene rpoB causing resistance to rifampin (Rifr) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global effects of rpoB mutations on metabolism, wild-type and 11 distinct congenic Rifr mutant strains of B. subtilis were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the Rifr mutants, including the utilization of novel substrates previously unknown in B. subtilis, such as gentiobiose, β-methyl-d-glucoside, and d-psicose. The results indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in B. subtilis.

scholarly journals The Global Regulator Spx Functions in the Control of Organosulfur Metabolism in Bacillus subtilis

2006 ◽  
Vol 188 (16) ◽  
pp. 5741-5751 ◽  
Author(s):  
Soon-Yong Choi ◽  
Dindo Reyes ◽  
Montira Leelakriangsak ◽  
Peter Zuber
Keyword(s):  
Bacillus Subtilis ◽  
In Vitro Transcription ◽  
Sulfur Source ◽  
Global Regulator ◽  
Control Of Gene Expression ◽  
Α Subunit ◽  
Cysteine Synthesis ◽  
Operon Expression ◽  
State Growth

ABSTRACT Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis. Its target is RNA polymerase, where it contacts the α 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, steady-state growth.

scholarly journals An endogenous dAMP ligand in Bacillus subtilis class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP

2018 ◽  
Vol 115 (20) ◽  
pp. E4594-E4603 ◽  
Author(s):  
Mackenzie J. Parker ◽  
Ailiena O. Maggiolo ◽  
William C. Thomas ◽  
Albert Kim ◽  
Steve P. Meisburger ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Dependent Manner ◽  
Α Subunit ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Dna Replication And Repair ◽  
Class Ib

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme’s quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5′-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.

scholarly journals Analysis of Temporal Gene Expression during Bacillus subtilis Spore Germination and Outgrowth

2007 ◽  
Vol 189 (9) ◽  
pp. 3624-3634 ◽  
Author(s):  
Bart J. F. Keijser ◽  
Alex Ter Beek ◽  
Han Rauwerda ◽  
Frank Schuren ◽  
Roy Montijn ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dependent Manner ◽  
Temporal Gene Expression ◽  
Bacillus Subtilis Spore ◽  
Physiological Processes ◽  
Genome Wide ◽  
Extracellular Metabolites ◽  
A Genome ◽  
Spore Outgrowth

ABSTRACT Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome.

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