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 Autoinduction of Bacillus subtilis phoPR Operon Transcription Results from Enhanced Transcription from EσA- and EσE-Responsive Promoters by Phosphorylated PhoP

2004 ◽  
Vol 186 (13) ◽  
pp. 4262-4275 ◽  
Author(s):  
Salbi Paul ◽  
Stephanie Birkey ◽  
Wei Liu ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Phosphate Starvation ◽  
Growth Conditions ◽  
In Vitro Transcription ◽  
Phosphate Deficiency ◽  
Pho Regulon ◽  
Environmental Controls ◽  
Rna Polymerase Holoenzyme

ABSTRACT The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted EσA, EσB, and EσE holoenzymes identified PA4 and PA3 as EσA promoters and PE2 as an EσE promoter. Phosphorylated PhoP (PhoP∼P) enhanced transcription from each of these promoters. EσB was sufficient for in vitro transcription of the PB1 promoter. P5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoP∼P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP∼P at an EσE promoter. Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.

scholarly journals Transcription Regulation of ezrA and Its Effect on Cell Division of Bacillus subtilis

2004 ◽  
Vol 186 (17) ◽  
pp. 5926-5932 ◽  
Author(s):  
Kuei-Min Chung ◽  
Hsin-Hsien Hsu ◽  
Suresh Govindan ◽  
Ban-Yang Chang
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Critical Concentration ◽  
In Vitro Transcription ◽  
Ring Formation ◽  
Negative Regulator ◽  
Cell Length ◽  
Intracellular Level ◽  
Z Ring

ABSTRACT The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA 96:9642-9647, 1999). We have studied the regulation of ezrA expression during the growth of B. subtilis and its effects on the intracellular level of EzrA as well as the cell length of B. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping σA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified. P1, supposed to be an extended −10 promoter, was responsible for most of the ezrA expression during the growth of B. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA in B. subtilis. More importantly, the cell length and percentage of filamentous cells of B. subtilis were significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth of B. subtilis, despite serving as a negative regulator for Z-ring formation.

Mutational studies of archaeal RNA polymerase and analysis of hybrid RNA polymerases

2009 ◽  
Vol 37 (1) ◽  
pp. 18-22 ◽  
Author(s):  
Michael Thomm ◽  
Christoph Reich ◽  
Sebastian Grünberg ◽  
Souad Naji
Keyword(s):  
Complex Formation ◽  
Structural Similarity ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Hyperthermophilic Archaea ◽  
Active Centre ◽  
Α Subunit ◽  
Recent Success ◽  
Open Complex Formation

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure–function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the α subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the ΔH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

scholarly journals Hybrid Bordetella pertussis-Escherichia coli RNA Polymerases: Selectivity of Promoter Activation

1998 ◽  
Vol 180 (6) ◽  
pp. 1567-1569 ◽  
Author(s):  
Pierre Steffen ◽  
Agnes Ullmann
Keyword(s):  
Escherichia Coli ◽  
Bordetella Pertussis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Α Subunit ◽  
E Coli ◽  
Transcription System ◽  
Catabolite Gene Activator Protein ◽  
Transcription Activators

ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

scholarly journals Role of PhoP∼P in Transcriptional Regulation of Genes Involved in Cell Wall Anionic Polymer Biosynthesis in Bacillus subtilis

1998 ◽  
Vol 180 (15) ◽  
pp. 4007-4010 ◽  
Author(s):  
Ying Qi ◽  
F. Marion Hulett
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Acid Synthesis ◽  
In Vitro Transcription ◽  
Anionic Polymer ◽  
Transcription System ◽  
Teichuronic Acid

ABSTRACT tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation oftuaA and the repression of tagA andtagD. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.

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 Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

2002 ◽  
Vol 184 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
In Vitro Transcription ◽  
Dna Binding Domain ◽  
Promoter Activation ◽  
Transcription Activators

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

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