Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma 32.

Escherichia coli K-12 strain 285c contains a short deletion mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase. The sigma 70 protein encoded by this allele (rpoD285) unstable, and this instability leads to temperature-sensitive growth. Pseudorevertants of 285c that can grow at high temperature contain mutations in the rpoH gene (encoding the heat shock sigma factor sigma 32), and their mutant sigma 70 proteins have increased stability. We characterized the alterations in three of these rpoH alleles. rpoH111 was a point mutation resulting in a single amino acid substitution. rpoH107 and rpoH113, which are known to be incompatible with rpoD+, altered the restriction map of rpoH. rpoH113 was deleted for 72 base pairs of the rpoH gene yet retained some sigma 32 activity. rpoH107 had two IS1 elements that flanked an unknown DNA segment of more than 6.4 kilobases inserted in the rpoH promoter region. The insertion decreased the amount of rpoH mRNA to less than 0.5% of the wild-type level at 30 degrees C. However, the mRNA from several heat shock promoters was decreased only twofold, suggesting that the strain has a significant amount of sigma 32.     

Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis.

The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.     

Overproducing the Bacillus subtilis mother cell sigma factor precursor, Pro-sigma K, uncouples sigma K-dependent gene expression from dependence on intercompartmental communication.

During sporulation of Bacillus subtilis, proteolytic activation of pro-sigma K and ensuing sigma K-dependent gene expression normally require the activity of many sporulation gene products. We report here that overproducing pro-sigma K at the onset of sporulation substantially uncouples sigma K-dependent gene expression from its normal dependency. Overproducing pro-sigma K in strains with a mutation in spoIIIG, spoIIIA, spoIIIE, or spoIVB partially restored sigma K-dependent gene expression in the mother cell and resulted in accumulation of a small amount of polypeptide that comigrated with sigma K, but these mutants still failed to form spores. In contrast, sporulation of spoIVF mutants was greatly enhanced by pro-sigma K overproduction. The products of the spoIVF operon are made in the mother cell and normally govern pro-sigma K processing, but overproduction of pro-sigma K appears to allow accumulation of a small amount of sigma K, which is sufficient to partially restore mother cell gene expression and spore formation. This spoIVF-independent mechanism for processing pro-sigma K depends on sigma E, an earlier-acting mother cell-specific sigma factor. The spoIIID gene, which encodes a mother cell-specific DNA-binding protein that is normally required for pro-sigma K production, was shown to be required for efficient pro-sigma K processing as well. bof (bypass of forespore) mutations bypassed this requirement for spoIIID, suggesting that SpoIIID is less directly involved in pro-sigma K processing than are spoIVF gene products. However, bof spoIIID double mutants overproducing pro-sigma K still failed to sporulate, indicating that SpoIIID serves another essential role(s) in sporulation in addition to its multiple roles in the production of sigma K.     

The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.     

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.     

A new mutation rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants

Summary We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon transfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45°C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9° C lower than does the wild type.The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E. coli RNA polymerase.     

Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase.

Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.     

Cloning and characterization of a sigma 70 homologue gene, sigA from Corynebacterium ammoniagenes

A sigma 70-like gene, sigA, has been identified from Corynebacterium ammoniagenes. The sigA gene encodes a polypeptide of 467 amino acids with a calculated molecular mass of 52036 Da. The deduced amino acid sequence preserves the common motifs of the primary sigma factors and shows very high similarity to those of SigA (sigmaA) homologues from high G+C Gram-positive bacteria, which suggest that the sigA gene encodes the primary sigma factor. The sigA gene is transcribed as a monocistronic mRNA of 2 kb and its mRNA occurs during the exponential growth phase and decays rapidly on entry into the stationary phase. The open reading frame encoding polyphosphate glucokinase-like protein is closely linked to the sigA gene.