Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in Bacillus subtilis (original) (raw)
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CcpA forms complexes with CodY and RpoA in Bacillus subtilis
FEBS Journal, 2012
The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the a-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose, whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (aNTD) and C-terminal domain (aCTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the aNTD and the aCTD were detected by surface plasmon resonance. The K D values of complexes of CcpA or CodY with the aNTD or the aCTD are in the range 5-8 lM. CcpA and CodY form a loose complex with a K D of 60 lM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.
Multiple regulatory sites in the Bacillus subtilis citB promoter region
Journal of Bacteriology, 1990
The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate. It is derepressed when either of these nutrient sources becomes limiting. Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription. In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression. Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression. By gel mobility shift assays, it was shown that at least one protein in extracts of B. subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively. A second sequence loc...
Molecular Microbiology, 2003
acs encodes acetyl-coenzyme A synthetase, a highaffinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acs P2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP-dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti-activators of CRP, each working independently at different times during growth to set the levels of CRP-dependent acs transcription.
Complex Regulation of the Bacillus subtilis Aconitase Gene
Journal of Bacteriology, 2003
The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase ( citB ) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter regi...
Microbiology, 2010
Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid biosynthesis. The molecular mechanisms of regulation of the acpP structural gene, as well as the function of its gene product, are poorly characterized in Bacillus subtilis and other Gram-positive organisms. Here, we report that transcription of acpP takes place from two different promoters: PfapR and PacpP. Expression of acpP from PfapR is coordinated with a cluster of genes involved in lipid synthesis (the fapR operon); the operon consists of fapR-plsX-fabD-fabG-acpP. PacpP is located immediately upstream of the acpP coding sequence, and is necessary and sufficient for normal fatty acid synthesis. We also report that acpP is essential for growth and differentiation, and that ACP localizes in the mother-cell compartment of the sporangium during spore formation. These results provide the first detailed characterization of the expression of the ACP-encoding gene in a Gram-positive bacterium, and highlight the importance of this protein in B. subtilis physiology.
Expression of Two Escherichia coli Acetyl-CoA Carboxylase Subunits Is Autoregulated
Journal of Biological Chemistry, 2003
Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid biosynthesis, the synthesis of malonyl-CoA from acetyl-CoA using ATP and bicarbonate. In Escherichia coli and most other bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Prior work from this laboratory showed that the in vivo expression of the accBC operon had a strikingly nonlinear response to gene copy number (Li, S.-J, and Cronan, J. E., Jr. (1993) J. Bacteriol. 175, 332-340) in that the presence of 50 or more copies of the accBC operon resulted in only a 2-3-fold increase in AccB and AccC. We now report that AccB functions to negatively regulate transcription of the accBC operon. Expression of a chimeric protein consisting of the N terminus of E. coli AccB and the C-terminal bioinylation domain of Bacillus subtilis AccB down-regulated transcription of the E. coli accBC operon. A truncated form of AccB consisting of the N-terminal 68 amino acids of E. coli AccB was sufficient to negatively regulate the accBC operon. In vivo bypass of acetyl-CoA carboxylase activity by expression of a malonyl-CoA synthase from Rhizobium trifolii allowed construction of strain deleted for the accA and accB genes. Unexpectedly, the ⌬accB mutation could not be resolved from the ⌬accA mutation. Transcription of the accBC operon in the ⌬accB ⌬accA strain continued well into stationary phase under growth conditions that normally result in greatly decreased transcription. These data support a model in which AccB acts as an autoregulator of accBC operon transcription.
Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity
PLoS ONE, 2010
Evidence suggesting that eukaryotes and archaea use reversible N e -lysine (N e -Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of N e -Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD + -dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsB Ac ), demonstrating that N e -Lys acetylation of RcsB is reversible. Analysis of RcsB Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N e -Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.
Molecular Microbiology, 2005
The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having + 5 bp and + 10 bp insertions between the two ToxT binding sites indicate that both binding sites are occupied by ToxT regardless of their positions relative to each other. Based on these results, we propose that ToxT binds independently to two DNA sites between acfA and acfD to activate transcription of both genes.
Journal of Molecular Biology, 1997
and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe puri®cation of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Speci®c interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre* sites, one of which overlaps the À35 box of the xyl promoter. Binding to cre* depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre* in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The CcpA dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the ®rst member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.