Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

ABSTRACT: The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

SUBMITTER: Bower S

PROVIDER: S-EPMC176921 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

Bower S S Perkins J J Yocum R R RR Serror P P Sorokin A A Rahaim P P Howitt C L CL Prasad N N Ehrlich S D SD Pero J J

Journal of bacteriology 19950501 9

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence simila ...[more]

PMID: 7730294

Similar Datasets

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

Project description:A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

| S-EPMC178169 | biostudies-other

Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1.

Project description:The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE. Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-beta-D-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family. yueB encodes a membrane protein of approximately 120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA. Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained. Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains. Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate. Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.

| S-EPMC532427 | biostudies-literature

Identification of the Bacillus subtilis pur operon repressor.

Project description:Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

| S-EPMC41358 | biostudies-other

Cloning and characterization of the groESL operon from Bacillus subtilis.

Project description:The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

| S-EPMC206107 | biostudies-other

Molecular cloning and characterization of fengycin synthetase gene fenB from Bacillus subtilis.

Project description:A fengycin synthetase gene, fenB, has been cloned and sequenced. The protein (FenB) encoded by this gene has a predicted molecular mass of 143.6 kDa. This protein was overexpressed in Escherichia coli and was purified to near homogeneity by affinity chromatography. Experimental results indicated that the recombinant FenB has a substrate specificity toward isoleucine with an optimum temperature of 25 degrees C, an optimum pH of 4.5, a Km value of 922 microM, and a turnover number of 236 s(-1). FenB also consists of a thioesterase domain, suggesting that this protein may be involved in the activation of the last amino acid of fengycin.

| S-EPMC107026 | biostudies-literature

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

Project description:The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.

| S-EPMC178230 | biostudies-other

Purification and characterization of the DeoR repressor of Bacillus subtilis.

Project description:Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.

| S-EPMC101877 | biostudies-literature

Characterization of DinR, the Bacillus subtilis SOS repressor.

Project description:In Bacillus subtilis, exposure to DNA damage and the development of natural competence lead to the induction of the SOS regulon. It has been hypothesized that the DinR protein is the cellular repressor of the B. subtilis SOS system due to its homology to the Escherichia coli LexA transcriptional repressor. Indeed, comparison of DinR and its homologs from gram-negative and -positive bacteria revealed conserved structural motifs within the carboxyl-terminal domain that are believed to be important for autocatalysis of the protein. In contrast, regions within the DNA binding domain were conserved only within gram-negative or -positive genera, which possibly explains the differences in the sequence specificities between gram-negative and gram-positive SOS boxes. The hypothesis that DinR is the repressor of the SOS regulon in B. subtilis has been tested through overexpression, purification, and characterization of the DinR protein. Like E. coli LexA, B. subtilis DinR undergoes an autocatalytic reaction at alkaline pH at a siscile Ala91-Gly92 bond. The cleavage reaction can also be mediated in vitro under more physiological conditions by the E. coli RecA protein. By using electrophoretic mobility shift assays, we demonstrated that DinR interacts with the previously characterized SOS box of the B. subtilis recA gene, but not with sequences containing single base pair mutations within the SOS box. Together, these observations strongly suggest that DinR is the repressor of the SOS regulon in B. subtilis.

| S-EPMC178884 | biostudies-other

Gene cloning, transcriptional analysis, purification, and characterization of phenolic acid decarboxylase from Bacillus subtilis.

Project description:Bacillus subtilis displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic, p-coumaric, and caffeic acids. Based on DNA sequence homologies between the Bacillus pumilus ferulate decarboxylase gene (fdc) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484-4486, 1995) and the Lactobacillus plantarum p-coumarate decarboxylase gene (pdc) (J.-F. Cavin, L. Barthelmebs, and C. Diviès, Appl. Environ. Microbiol. 63:1939-1944, 1997), a DNA probe of about 300 nucleotides for the L. plantarum pdc gene was used to screen a B. subtilis genomic library in order to clone the corresponding gene in this bacterium. One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the pdc- and fdc-encoded enzymes, respectively. The PAD gene (pad) is transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The pad gene was overexpressed constitutively in Escherichia coli, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.

| S-EPMC106171 | biostudies-literature

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis.

Project description:The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.

| S-EPMC106281 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data