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J D Helmann

Publications and source records attributed to J D Helmann.

At least 55 records · Page 3Linked to original sources
PubMed1994

A promoter melting region in the primary sigma factor of Bacillus subtilis. Identification of functionally important aromatic amino acids.

Sigma factor (sigma) is a dissociable subunit of bacterial RNA polymerase that determines promoter recognition. It has been proposed that a cluster of highly conserved aromatic amino acids in bacterial sigma factors (region 2.3) defines a melting motif that functions in strand-separation during open complex formation. We demonstrate that many alterations in region 2.3 of the Bacillus subtilis sigma A protein specifically impair open complex formation. The region 2.3 mutations can be grouped in three classes: (1) mutations that do not significantly affect promoter recognition or melting; (2) mutations that lead to cold-sensitive transcription of linear templates; and (3) mutations that lead to little activity on linear templates but retain activity at high temperatures on supercoiled templates. RNA polymerase holoenzymes containing sigma factor melting mutants (classes 2 and 3) form predominantly closed complexes at 40 degrees C and are defective for RNA synthesis when initiation is rate-limiting. The melting defect of these mutant sigma factors is suppressed by template supercoiling, but further enhanced by inclusion of the auxiliary RNA polymerase subunit delta. Consequently, in the presence of the delta polypeptide, the mutant holoenzymes display cold-sensitive transcription on supercoiled templates: conditions which mimic the in vivo situation. A subset of these mutations also affects promoter selectivity, suggesting that region 2.3 may participate in both -10 recognition and DNA melting.

Amino Acid Sequence↗
PubMed1994

The Bacillus subtilis sigma D-dependent operon encoding the flagellar proteins FliD, FliS, and FliT.

During a genetic screen to identify metalloregulated loci in Bacillus subtilis, we isolated a Tn917-lacZ insertion in the second gene of an operon downstream of the flagellin (hag) gene. Sequence analysis indicates that this gene encodes a homolog of the enteric flagellar filament cap protein FliD. The fliD gene is followed by homologs of the fliS and fliT genes. Transcription of the fliD-lacZ fusion is sigma D dependent, with peak expression at the end of logarithmic-phase growth. Like other sigma D-dependent genes, expression of fliD-lacZ is greatly reduced by mutations in genes essential for assembly and function of the basal body and hook complex (class II functions). These results suggest that B. subtilis flagellar genes are organized in a hierarchy of gene expression similar to that found in enteric bacteria with hag and fliD as class III genes. Expression from the fliD operon promoter, but not the hag promoter, is repressed by iron, which suggests that the target of metalloregulation is the promoter rather than the sigma D protein.

Amino Acid Sequence↗
PubMed1994

Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting.

The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition.

Alleles↗
PubMed1994

Dual chemotaxis signaling pathways in Bacillus subtilis: a sigma D-dependent gene encodes a novel protein with both CheW and CheY homologous domains.

The alternative sigma factor, sigma D, activates the expression of genes required for chemotaxis and motility in Bacillus subtilis, including those encoding flagellin, hook-associated proteins, and the motor proteins. The sigma D protein is encoded in a large operon which also encodes the structural proteins for the basal body and homologs of the enteric CheW, CheY, CheA, and CheB chemotaxis proteins. We report the identification and molecular characterization of a novel chemotaxis gene, cheV. The predicted CheV gene product contains an amino-terminal CheW homologous domain linked to a response regulator domain of the CheY family, suggesting that either or both of these functions are duplicated. Transcription of cheV initiates from a sigma D-dependent promoter element both in vivo and in vitro, and expression of a cheV-lacZ fusion is completely dependent on sigD. Expression is repressed by nonpolar mutations in structural genes for the basal body, fliM or fliP, indicating that cheV belongs to class III in the B. subtilis flagellar hierarchy. The cheV locus is monocistronic and is located at 123 degrees on the B. subtilis genetic map near the previously defined cheX locus. A cheV mutant strain is motile but impaired in chemotaxis on swarm plates. Surprisingly, an insertion in the CheW homologous domain leads to a more severe defect than an insertion in the CheY homologous domain. The presence of dual pathways for chemotactic signal transduction is consistent with the residual signaling observed in previous studies of cheW mutants (D. W. Hanlon, L. Márques-Magaña, P. B. Carpenter, M. J. Chamberlin, and G. W. Ordal, J. Biol. Chem. 267:12055-12060, 1992).

Amino Acid Sequence↗
PubMed1994

Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs.

We have characterized mutants in a novel gene of Bacillus subtilis, cheV, which encodes a protein homologous to both CheW and CheY. A null mutant in cheV is only slightly defective in capillary and tethered cell assays. However, a double mutant lacking both CheV and CheW has a strong tumble bias, does not respond to addition of attractant, and shows essentially no accumulation in capillary assays. Thus, CheV and CheW appear in part to be functionally redundant. A strain lacking CheW and expressing only the CheW domain of CheV is chemotactic, suggesting that the truncated CheV protein retains in vivo function. We speculate that CheV and CheW function together to couple CheA activation to methyl-accepting chemotaxis protein receptor status and that possible CheA-dependent phosphorylation of CheV contributes to adaptation.

Bacillus subtilis↗
PubMed1993

Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions.

We have cloned two metal-regulated genes (mrgA and mrgC) from Bacillus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted sigma A-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.

Amino Acid Sequence↗
PubMed1992

Restoration of motility to an Escherichia coli fliA flagellar mutant by a Bacillus subtilis sigma factor.

The activation of additional promoter sites by production of an alternative sigma subunit for RNA polymerase is a common strategy for the coordinate regulation of gene expression. Many alternative sigma factors control genes for specialized, and often narrowly distributed, functions. For example, most of the alternative sigma factors in Bacillus subtilis control genes necessary for endospore formation. In contrast, the B. subtilis sigma D protein controls the expression of genes important for flagellar-based motility and chemotaxis, a form of locomotion very broadly distributed in the eubacteria. A homologous sigma factor, sigma F, controls a similar group of motility genes in the enteric bacteria. The conservation of both promoter specificity and genetic function in these two regulons allowed us to test the ability of a B. subtilis sigma factor to function within an Escherichia coli host. We demonstrate that expression of the B. subtilis sigD gene restores motility to an E. coli strain mutant in the fliA locus encoding the sigma F factor. This result suggests that the B. subtilis sigma D protein can bind to the E. coli core RNA polymerase to direct transcription initiation from at least four of the late operon promoters, thereby leading to the synthesis of flagellin, motor, and hook-associated proteins. Conversely, expression of sigma D protein in a normally chemotactic strain of E. coli (fliA+) leads to a hyperflagellated, nonchemotactic phenotype.

Bacillus subtilis↗
PubMed1992

The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite.

Octopine is released from crown gall tumors as a nutrient source and a signal molecule for the plant pathogen Agrobacterium tumefaciens. Some or all octopine-inducible genes are regulated by a protein called OccR. Primer extension analysis showed that OccR protein represses the occR gene and both represses and activates the occQ operon, which is divergently transcribed from occR. These promoters initiate transcription 46 bp apart. This regulatory system was reconstituted in vitro using purified OccR protein and Escherichia coli RNA polymerase. OccR binds with high affinity to a single site overlapping these promoters. Octopine shortens the DNAase I footprint of OccR and increases the gel mobility of OccR-DNA complexes by relaxing an OccR-incited DNA bend.

Agrobacterium tumefaciens↗
PubMed1991

Alternative sigma factors and the regulation of flagellar gene expression.

Synthesis of bacterial flagella and the accompanying array of chemotaxis receptors and transducers represents a major commitment of energy and resources for a growing bacterial cell and is subject to numerous levels of regulation. Genes for flagellar and chemotaxis proteins are expressed in a complex transcriptional cascade. This regulatory hierarchy acts to ensure that the highly expressed filament structural protein, flagellin, is synthesized only after a prerequisite set of structural proteins has been expressed and properly assembled. Recent evidence suggests that many bacteria utilize an alternative sigma (sigma) subunit, similar in specificity to the Bacillus subtilis sigma 28 protein, to direct transcription of flagellin, chemotaxis and motility genes. In Caulobacter crescentus and Campylobacter spp., both a sigma 54-like factor and a sigma 28-like factor participate in the transcription of flagellar and chemotaxis genes. Conversely, a sigma 28-like factor controls non-motility functions in at least one non-flagellated organism.

Base Sequence↗
PubMed1990

The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer.

Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.

Amino Acid Sequence↗
PubMed1990

Studies of sigma D-dependent functions in Bacillus subtilis.

Gene expression in Bacillus subtilis can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. One such factor, sigma D (sigma 28), is expressed during vegetative growth and has been implicated in the transcription of a regulon of genes expressed during exponential growth and the early stationary phase. We have studied several functions related to flagellar synthesis and chemotaxis in B. subtilis strains in which sigma D is missing or is present at reduced levels. Previous studies showed that a null mutant, which contains a disrupted copy of the sigma D structural gene (sigD), fails to synthesize flagellin and grows as long filaments. We now show that these defects are accompanied by the lack of synthesis of the methyl-accepting chemotaxis proteins and a substantial decrease in two autolysin activities implicated in cell separation. A strain containing an insertion upstream of the sigD gene that reduces the level of sigma D protein grew as short chains and was flagellated but was impaired in chemotaxis and/or motility. This reduced level of sigma D expression suggests that the sigD gene may be part of an operon. A strain containing an insertion downstream of the sigD gene expressed nearly wild-type levels of sigma D protein but was also impaired in chemotaxis and/or motility, suggesting that genes downstream of sigD may also be involved in these functions. Genetic experiments demonstrate that sigD is allelic to the flaB locus, which was initially isolated as a locus affecting flagellin expression (G. F. Grant and M. I. Simon, J. Bacteriol. 99:116-124, 1969).

Alleles↗
PubMed1989

The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis.

In the mycelial prokaryote S. coelicolor, whiG is a gene dispensable for growth but needed for the earliest stages of spore formation in aerial hyphae. Nucleotide sequencing indicates that whiG encodes an RNA polymerase sigma factor highly similar to the motility sigma factor (sigma 28) of B. subtilis. High copy number of an intact whiG gene caused sporulation in vegetative hyphae that are usually fated to lyse without sporulating. However, the introduction of many copies of a sigma 28-dependent promoter from B. subtilis into S. coelicolor reduced sporulation, suggesting partial sequestration of the whiG gene product by the foreign promoter sequences. We propose that the level of whiG sigma factor is crucial in determining the developmental fate of hyphae.

Amino Acid Sequence↗
PubMed1989

Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains.

Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a-r-) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a-r+) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variably deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data, taken with the results in the preceding paper (Shewchuk et al., 1989), support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation.

Amino Acid Sequence↗
PubMed1989

Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons.

We report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator region in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the Bacillus subtilis sigma 28 factor.

RNA polymerase preparations isolated from vegetatively growing Bacillus subtilis cells contain the core subunits beta, beta', and alpha, together with multiple sigma factors and other core-associated polypeptides such as delta, omega 1, and omega 2. We have developed an improved, large-scale purification procedure that yields RNA polymerase fractions enriched in both the sigma 28 and delta proteins. These fractions have been used to isolate sigma 28 protein for biochemical characterization and for preparation of highly specific anti-sigma 28 antisera. The amino acid composition of purified sigma 28 protein and the amino acid sequences of tryptic peptide fragments have been determined. Anti-sigma 28 antisera specifically inhibit transcription by the purified sigma 28 -dependent RNA polymerase, yet do not affect transcription by sigma 43 -dependent RNA polymerase. Immunochemical analysis confirms that the sigma 28 protein copurifies with total RNA polymerase activity through the majority of the purification procedure and allows the steps when sigma 28 protein is lost to be identified and optimized. Immunochemical techniques have also been used to monitor the structure and abundance of the sigma 28 protein in vivo. A single form of antibody-reactive protein was detected by two-dimensional gel electrophoresis-isoelectric focusing. Its abundance corresponds to a maximal content of 220 molecules of sigma 28 per B. subtilis cell during late-logarithmic-phase growth.

Amino Acid Sequence↗
PubMed1988

Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene.

Bacillus subtilis contains multiple forms of RNA polymerase holoenzyme, distinguished by the presence of different specificity determinants known as sigma factors. The sigma 28 factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the sigma 28 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the sigma 28 structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the sigma 28 gene was determined, and the cloned sigma 28 gene was used to construct a mutant strain which does not express the sigma 28 protein. This strain also failed to synthesize flagellin protein and grew as long filaments. The predicted sigma 28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The sigma 28 protein sequence was similar to that of other sequenced sigma factors and to the flbB gene product of Escherichia coli. Since the flbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that sigma 28 functions to regulate flagellar synthesis in B. subtilis.

Amino Acid Sequence↗