Biomedical subjects

L V Wray

Publications and source records attributed to L V Wray.

At least 19 recordsLinked to original sources

PubMed2001

Bacillus subtilis glutamine synthetase controls gene expression through a protein-protein interaction with transcription factor TnrA.

Bacillus subtilis TnrA, a global regulator of transcription, responds to nitrogen availability, but the specific signal to which it responds has been elusive. Genetic studies indicate that glutamine synthetase is required for the regulation of TnrA activity in vivo. We report here that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA and blocks the DNA binding activity of TnrA. Mutations in the tnrA gene (tnrA(C)) that allow constitutive high level expression of tnrA-activated genes were isolated and characterized. Feedback-inhibited glutamine synthetase had a significantly reduced ability to block the in vitro DNA binding by three of the TnrA(C) proteins. Thus, glutamine synthetase, an enzyme of central metabolism, directly interacts with and regulates the DNA binding activity of TnrA.

Amino Acid Sequence↗

PubMed2000

Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor.

The Bacillus subtilis nitrogen regulatory protein TnrA was purified and its interaction with the nrgAB regulatory region examined. The TnrA protein activates transcription from the nrgAB promoter in vitro. DNase I footprinting and methylation protection experiments demonstrated that TnrA binds to an inverted repeat, upstream of the -35 region of the nrgAB promoter. Gel mobility retardation assays were used to determine the affinity of TnrA for its DNA-binding site. The equilibrium dissociation binding constant for the interaction of TnrA with the nrgAB promoter fragment was 7.7 nM under the conditions used here. Mutations in the TnrA consensus sequence that reduce nrgAB expression in vivo were found to reduce significantly the in vitro affinity for TnrA. An A+T rich region located upstream of the TnrA-binding site was found to be necessary for optimal transcriptional activation. A mutant protein, TnrA(HTH), was constructed in which the putative helix-turn-helix DNA-binding motif was altered by exchanging two arginine residues for alanine residues. The TnrA(HTH) protein was unable to activate the in vivo expression of nrgAB and had an in vitro affinity for the nrgAB promoter that was significantly lower than that of the wild-type protein.

Amino Acid Sequence↗

PubMed2000

Role of TnrA in nitrogen source-dependent repression of Bacillus subtilis glutamate synthase gene expression.

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

Bacillus subtilis↗

PubMed1999

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis.

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

Bacillus subtilis↗

PubMed1998

Transcription-repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons.

A Bacillus subtilis mutant that partially relieves carbon catabolite repression (CCR) of the hut operon was isolated by transposon mutagenesis. Characterization of this mutant revealed that the transposon had inserted into the gene, mfd, that encodes transcription-repair coupling factor. The Mfd protein is known to promote strand-specific DNA repair by displacing RNA polymerase stalled at a nucleotide lesion and directing the (A)BC excinuclease to the DNA damage site. A set of transcriptional lacZ fusions was used to demonstrate that the mfd mutation relieves CCR of hut and gnt expression at the cis-acting cre sequences located downstream of the transcriptional start site but does not affect CCR at sites located at the promoters. CCR of the amyE and bglPH genes, which contain cre sequences that overlap their promoters, is not altered by the mfd mutation. These results support a model in which the Mfd protein displaces RNA polymerase stalled at downstream cre sites that function as transcriptional roadblocks and reveal a new role for Mfd in cellular physiology.

Artificial Gene Fusion↗

PubMed1998

Mutational analysis of the TnrA-binding sites in the Bacillus subtilis nrgAB and gabP promoter regions.

Transcription of the Bacillus subtilis nrgAB promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN7TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated nrgAB, gabP P2, and nas promoters. Oligonucleotide-directed mutagenesis of the nrgAB promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression. Mutations that alter the relative distance between the two half-sites of the nrgAB TnrA site abolish nitrogen regulation of nrgAB expression. Spacer mutations that change the relative distance between the TnrA site and -35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the gabP P2 TnrA site showed that this sequence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, had a 625-fold-higher affinity for the wild-type nrgAB promoter DNA than for a mutated nrgAB promoter DNA fragment that is unable to activate nrgAB expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate nrgAB expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.

Bacillus subtilis↗

PubMed1998

Expression of the Bacillus subtilis acsA gene: position and sequence context affect cre-mediated carbon catabolite repression.

In Bacillus subtilis, carbon catabolite repression (CCR) of many genes is mediated at cis-acting carbon repression elements (cre) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor (mfd) partially relieve CCR at cre sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at cre sites which act as transcriptional roadblocks. Although the acsA cre is centered 44.5 bp downstream of the acsA transcriptional start site, CCR of acsA expression is not affected by an mfd mutation. When the acsA cre is centered 161.5 bp downstream of the transcriptional start site for the unregulated tms promoter, CCR is partially relieved by the mfd mutation. Since CCR mediated at an acsA cre centered 44.5 bp downstream of the tms start site is not affected by the mfd mutation, the inability of Mfd to modulate CCR of acsA expression most likely results from the location of the acsA cre. Higher levels of CCR were found to occur at cre sites flanked by A+T-rich sequences than at cre sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp cre consensus sequence participate in the formation of the CcpA catabolite repression complex at cre sites. Examination of CCR of acsA expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.

Bacillus subtilis↗

PubMed1997

The Bacillus subtilis ureABC operon.

The Bacillus subtilis ureABC operon encodes homologs of the three subunits of urease enzymes of the family Enterobacteriaceae. Disruption of ureC prevented utilization of urea as a nitrogen source and resulted in a partial growth defect in minimal medium containing limiting amounts of arginine or allantoin as the sole nitrogen source.

Allantoin↗

PubMed1997

Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H.

Expression of urease, which is encoded by the ureABC operon, is regulated in response to nitrogen availability in Bacillus subtilis. Three ureABC promoters were identified in primer extension experiments and by examination of beta-galactosidase expression from ure-lacZ fusions. P1, a low-level constitutive promoter, lies immediately upstream of ureA. The P2 promoter is transcribed by the E sigmaH form of RNA polymerase and initiates transcription 270 bp upstream of the ureA start codon. The transcriptional start site for the sigmaA-dependent P3 promoter is located 839 bp upstream of the ureA start codon. To identify transcription factors that control ureABC expression, regulation of the P2 and P3 promoters was examined in wild-type and mutant strains. During rapid growth in minimal medium containing glucose and amino acids, CodY represses expression of the P2 and P3 promoters 30- and 60-fold, respectively. TnrA activates expression of the P3 promoter 10-fold in nitrogen-limited cells, while GlnR represses transcription from the P3 promoter 55-fold during growth on excess nitrogen. Expression of the ureABC operon increases 10-fold at the end of exponential growth in nutrient sporulation medium. This elevation in expression results from the relief of CodY-mediated repression during exponential growth and increased sigmaH-dependent transcription during stationary phase.

Bacillus subtilis↗

PubMed1996

TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis.

Expression of the Bacillus subtilis nrgAB operon is derepressed during nitrogen-limited growth. We have identified a gene, tnrA, that is required for the activation of nrgAB expression under these growth conditions. Analysis of the DNA sequence of the tnrA gene revealed that it encodes a protein with sequence similarity to GlnR, the repressor of the B. subtilis glutamine synthetase operon. The tnrA mutant has a pleiotropic phenotype. Compared with wild-type cells, the tnrA mutant is impaired in its ability to utilize allantoin, gamma-aminobutyrate, isoleucine, nitrate, urea, and valine as nitrogen sources. During nitrogen-limited growth, transcription of the nrgAB, nasB, gabP, and ure genes is significantly reduced in the tnrA mutant compared with the levels seen in wild-type cells. In contrast, the level of glnRA expression is 4-fold higher in the, tnrA mutant than in wild-type cells during nitrogen restriction. The phenotype of the tnrA mutant indicates that a global nitrogen regulatory system is present in B. subtilis and that this system is distinct from the Ntr regulatory system found in enteric bacteria.

Amino Acid Sequence↗

PubMed1996

Expression of the Bacillus subtilis gabP gene is regulated independently in response to nitrogen and amino acid availability.

Expression from the Bacillus subtilis nrg-21 locus Increases 26-fold during nitrogen-limited growth. The DNA corresponding to this locus was cloned and sequenced. The nucleotide sequence revealed a gene that could encode a protein with sequence similarity to the Escherichia coll gamma-aminobutyric acid (GABA) permease. A transposon insertion in this locus eliminated the uptake of GABA and severely inhibited the utilization of GABA as a nitrogen source. Primer extension analysis revealed that the B. subtilis gabP gene was transcribed from two overlapping promoters. Transcription from the P1 promoter was repressed during growth in the presence of amino acids. The product of the codY gene proved to be required for this repression. Transcription from the P2 promoter increased during nitrogen-limited growth and was dependent upon the product of the tnrA gene. Deletion analysis revealed that activation of the P2 promoter during nitrogen-limited growth requires a nucleotide sequence located upstream of its -35 region. Regulation of gabP expression by the CodY and TnrA regulatory systems, which respond to different physiological signals, allows for a wide range of gabP expression during growth on various nitrogen sources.

Amino Acids↗

PubMed1994

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.

Amino Acid Sequence↗

PubMed1994

Analysis of Bacillus subtilis hut operon expression indicates that histidine-dependent induction is mediated primarily by transcriptional antitermination and that amino acid repression is mediated by two mechanisms: regulation of transcription initiation and inhibition of histidine transport.

Expression of the Bacillus subtilis hut operon is induced by histidine and subject to regulation by carbon catabolite repression and amino acid repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify the cis-acting sites required for histidine induction and amino acid repression. Histidine induction was found to be primarily mediated by transcriptional antitermination at a palindromic sequence located immediately downstream of the first structural gene in the hut operon, hutP. High levels of histidine induction were observed only in hut-lacZ fusions which contained this palindromic sequence. The hutC1 mutation, which results in constitutive expression of the hut operon, was sequenced and found to contain a GC to TA transversion located within the stem-loop structure. Transcription of hut DNA in vitro revealed that the palindromic structure functions as a transcriptional terminator with wild-type hut DNA but not with hutC1 DNA. Two sites were found to be involved in amino acid repression of hut expression: (i) an operator, hutOA, which lies downstream of the hut promoter, and (ii) the hut terminator. The rate of [14C]histidine uptake in amino acid-grown cells was sixfold lower than that seen in cells grown without amino acids. Thus, inhibition of histidine transport in amino acid-grown cells indirectly regulates hut expression by interfering with histidine induction at the hut terminator.

Bacillus subtilis↗

PubMed1994

Catabolite repression of the Bacillus subtilis hut operon requires a cis-acting site located downstream of the transcription initiation site.

Expression of the Bacillus subtilis hut operon is subject to regulation by catabolite repression. A set of hut-lacZ transcriptional fusions was constructed and used to identify two cis-acting sites involved in catabolite repression. The hutOCR1 operator site lies immediately downstream of the hut promoter and weakly regulates hut expression in response to catabolite repression. The downstream hutOCR2 operator site lies within the hutP gene, between positions +203 and +216, and is required for wild-type levels of catabolite repression. Both the hutOCR1 and hutOCR2 operators have sequence similarity to the sites which mediate catabolite repression of several other B. subtilis genes. Two mutations which relieve catabolite repression of hut expression were found to alter the nucleotide sequence of the hutOCR2 operator. Catabolite repression of hut expression was partially relieved in strains containing the ccpA mutation but not in strains containing either the pai or hpr mutation.

Aldose-Ketose Isomerases↗

PubMed1994

Modulation of Bacillus subtilis catabolite repression by transition state regulatory protein AbrB.

The first enzyme of the Bacillus subtilis histidine-degradative (hut) pathway, histidase, was expressed at higher levels during the onset of the stationary growth phase in nutrient sporulation medium in early-blocked sporulation mutants (spo0A) than in wild-type strains. Histidase expression was also elevated in spo0A mutant cultures compared with wild-type cultures during the logarithmic growth phase in minimal medium containing slowly metabolized carbon sources. Histidase expression was not derepressed in spo0A abrB mutant cultures under these growth conditions, suggesting that the AbrB protein is responsible for the derepression of histidase synthesis seen in spo0A mutant cultures. spo0A mutants contain higher levels of the AbrB protein than do wild-type strains because the Spo0A protein represses AbrB expression. A direct correlation between the levels of abrB transcription and histidase expression was found in spo0A mutant cultures. The hutOCR2 operator, which is required for wild-type regulation of hut expression by catabolite repression, was also required for AbrB-dependent derepression of hut expression in spo0A mutants. Purified AbrB protein bound to the hutOCR2 operator in vitro, suggesting that AbrB protein alters hut expression by competing with the hut catabolite repressor protein for binding to the hutOCR2 site. During the logarithmic growth phase in media containing slowly metabolized carbon sources, the expression of several other enzymes subject to catabolite repression was elevated in spo0A mutants but not in spo0A abrB mutants. This suggests that the AbrB protein acts as a global modulator of catabolite repression during carbon-limited growth.

Arabinose↗

PubMed1993

The Streptomyces coelicolor glnR gene encodes a protein similar to other bacterial response regulators.

The Streptomyces coelicolor glnR gene positively regulates the transcription of the glutamine synthetase-encoding glnA gene. The nucleotide sequence of a 1682-bp DNA segment containing glnR was determined. The deduced amino acid sequence of the GlnR protein was found to be similar to the sequence of several bacterial response regulators that are known to function as transcriptional activators. Primer extension analysis of glnR mRNA identified three transcriptional start points (tsp) upstream from the glnR coding sequence.

Amino Acid Sequence↗

PubMed1993

Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.

Amino Acids↗

PubMed1991

Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif.

We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.

Amino Acid Sequence↗