scholarly journals Effects of Bacillus subtilis sporulation regulatory protein SpoIIID on transcription by sigma K RNA polymerase in vivo and in vitro.

1995 ◽  
Vol 177 (7) ◽  
pp. 1888-1891 ◽  
Author(s):  
R Halberg ◽  
V Oke ◽  
L Kroos
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Regulatory Protein

scholarly journals The −10 Region Is a Key Promoter Specificity Determinant for the Bacillus subtilis Extracytoplasmic-Function ς Factors ςX and ςW

2001 ◽  
Vol 183 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Jian Qiu ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Context Effects ◽  
Promoter Strength ◽  
Binding Properties ◽  
Promoter Specificity ◽  
Dna Binding Properties

ABSTRACT Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in Bacillus subtilis which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ςX and ςW factors can be explained by overlapping specificity for the −10 region: ςX recognizes −10 elements with the sequence CGAC and ςW recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ςX-specific autoregulatory site (PX), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ςW autoregulatory site (PW) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ςX to the ςW regulon or, conversely, from the ςW to the ςX regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ςW.

scholarly journals Transcriptional Pausing in the Bacillus subtilis pyr Operon In Vitro: a Role in Transcriptional Attenuation?

2003 ◽  
Vol 185 (16) ◽  
pp. 4764-4771 ◽  
Author(s):  
Hesheng Zhang ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Rna Binding ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Stem Loop ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Transcriptional Pausing

ABSTRACT The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

scholarly journals Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis

2004 ◽  
Vol 186 (4) ◽  
pp. 1120-1128 ◽  
Author(s):  
K. A. Susanna ◽  
A. F. van der Werff ◽  
C. D. den Hengst ◽  
B. Calles ◽  
M. Salas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Complex Signal ◽  
Type I ◽  
Binding Characteristics ◽  
Α Subunits

ABSTRACT The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

scholarly journals Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3

2004 ◽  
Vol 186 (5) ◽  
pp. 1493-1502 ◽  
Author(s):  
Yinghua Chen ◽  
Wael R. Abdel-Fattah ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Phosphate Deficiency ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Α Helix

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoPH205A and PhoPV204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoPV202A and PhoPD203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoPV190A, PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoPW191A and PhoPR200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli.

scholarly journals Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

2007 ◽  
Vol 189 (9) ◽  
pp. 3348-3358 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Matthew Schau ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Regulatory Proteins ◽  
Regulation Of Respiration ◽  
Regulator Protein ◽  
Promoter Fusion

ABSTRACT The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

scholarly journals SpoIVB and CtpB Are Both Forespore Signals in the Activation of the Sporulation Transcription Factor σK in Bacillus subtilis

2007 ◽  
Vol 189 (16) ◽  
pp. 6021-6027 ◽  
Author(s):  
Nathalie Campo ◽  
David Z. Rudner
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Serine Proteases ◽  
Regulatory Protein ◽  
Signal Transduction Pathway ◽  
Proteolytic Activation ◽  
Proper Timing

ABSTRACT The proteolytic activation of the mother cell transcription factor pro-σK is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-σK processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-σK processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-σK processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of σK activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of σG is both necessary and sufficient for the proper timing of pro-σK processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein.

scholarly journals Interaction of Bacillus subtilis Fur (Ferric Uptake Repressor) with the dhb Operator In Vitro and In Vivo

1999 ◽  
Vol 181 (14) ◽  
pp. 4299-4307 ◽  
Author(s):  
Nada Bsat ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Metal Ion ◽  
Regulatory Region ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Metalloregulatory Proteins

ABSTRACT Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by ςA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the furgene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 μM but not at 5 μM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

scholarly journals Properties of Bacillus subtilis σA Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

2004 ◽  
Vol 186 (8) ◽  
pp. 2366-2375 ◽  
Author(s):  
Hsin-Hsien Hsu ◽  
Wei-Cheng Huang ◽  
Jia-Perng Chen ◽  
Liang-Yin Huang ◽  
Chai-Fong Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Activity ◽  
Transcription Activity ◽  
N Terminus ◽  
Σ Factor

ABSTRACT σ factors in the σ70 family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis σA, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σA and greatly reduced the transcription activity of the truncated σA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σA in RNA polymerase.

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