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.     

Multiple sigma factor genes in Brevibacterium lactofermentum: characterization of sigA and sigB.

Four rpoD hybridizing signals have been identified in the chromosome of Brevibacterium lactofermentum. Two rpoD-like genes, sigA and sigB, have been cloned and sequenced, and they encode principal sigma factors of the RNA polymerase. The deduced amino acid sequences of SigA and SigB showed very high similarities to those of Mycobacterium smegmatis MysA and MysB proteins, respectively, and also to those of HrdB proteins from different Streptomyces species. SigA and SigB maintain the conserved motifs of sigma 70-like principal sigma factors. sigB is closely linked to the dtxR gene (encoding a repressor of iron-regulated promoters homologous to the diphtheria toxin repressor from Corynebacterium diphtheriae.     

Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes.

The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.     

Circadian-regulated expression of a nuclear-encoded plastid sigma factor gene (sigA) in wheat seedlings.

The activity of a light-responsive psbD promoter in plastids is known to be regulated by a circadian clock. However, the mechanism of the circadian regulation of the psbD light-responsive promotor, which is recognized by an Escherichia coli-type RNA polymerase, is not yet known. We examined the time course of mRNA accumulation of two E. coli-type RNA polymerase subunit genes, sigA and rpoA, under a continuous light condition after 12 h light/12 h dark entrainment. Accumulation of the sigA mRNA was found to be regulated by a circadian clock, while rpoA mRNA did not show any significant oscillation throughout the experiment.     

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.     

Two chemosensory operons of Rhodobacter sphaeroides are regulated independently by sigma 28 and sigma 54

Rhodobacter sphaeroides has a complex chemosensory system, with several loci encoding multiple homologues of the components required for chemosensing in Escherichia coli. The operons cheOp2 and cheOp3 each encode complete pathways, and both are essential for chemosensing. The components of cheOp2 are predominantly localized to the cell pole, whereas those encoded by cheOp3 are predominantly targeted to a discrete cluster in the cytoplasm. Here we show that the expression of the two pathways is regulated independently. Overlapping promoters recognized by sigma(28) and sigma(70) RNAP holoenzyme transcribe cheOp2, whereas cheOp3 is regulated by one of the four sigma(54) homologues, RpoN3. The different regulation of these operons may reflect the need for balancing responses to extra- and intracellular signals under different growth conditions.     

Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence highly similar to the major sigma factors of Solibacter usitatus Ellin6076 and Geobacter sulfurreducens PCA. Purified hexahistidine-tagged RpoD migrates at approximately 70 kDa under SDS-PAGE conditions, which is consistent with the predicted MW of 69.2 kDa, and the gene product is immunoreactive with monoclonal antibodies specific for either bacterial RpoD proteins or the N-terminal histidine tag. A. capsulatum RpoD restored normal growth to E. coli strain CAG20153 under conditions that prevent expression of the endogenous rpoD. These results indicate we have cloned the gene encoding the A. capsulatum major sigma factor and the gene product is active in E. coli.     

The Staphylococcus aureus chromosomal gene plaC, identified by mutations amplifying plasmid pT181, encodes a sigma factor.

The Staphylococcus aureus chromosomal gene plaC, identified by mutations such as plaC1 that lead to the amplification of plasmid pT181, has been cloned and sequenced. The plaC gene encodes a protein with high similarity (79% identity) with the vegetative sigma factor of Bacillus subtilis, sigA, suggesting that it acts as an RNA polymerase sigma factor in S.aureus. The plaC1 mutation was found to be a C to T transition leading to a proline to serine substitution at amino acid residue 209 of the protein. In other sigma factors this region of the protein is involved in specific recognition of the -10 promoter sequence. The change in sigma factor activity due to this mutation is characterized by its strict specificity for a limited number of promoters and the rather high amplitude of the effect.     

A new putative sigma factor of Myxococcus xanthus.

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.