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.     

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.     

Expression of primary sigma factor (PSF) and PSF-like sigma factors in the cyanobacterium Synechocystis sp. strain PCC 6803

Large amounts of sigA mRNA, encoding the primary sigma factor (PSF) in Synechocystis sp. strain PCC 6803, accumulated under standard growth conditions, while stress conditions like heat or high salinity led to a rapid decrease in sigA mRNA content. The sigB, sigC, sigD, and sigE genes, encoding PSF-like sigma factors, were under strict physiological control.     

A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis.

Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.     

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.     

Evidence of Gene Conversion Events Between Paralogous Sequences Produced by Tetraploidization in Salmoninae Fish

We investigated the occurrence of gene conversions between paralogous sequences of Salmoninae derived from ancestral tetraploidization and their effect on the evolutionary history of DNA sequences. A microsatellite with long flanking regions (750 bp) including both coding and noncoding sequences was analyzed. Microsatellite size polymorphism was used to detect the alleles of both paralogous counterparts and infer linkage arrangement between loci. DNA sequencing of seven Salmoninae species revealed that paralogous sequences were highly differentiated within species, especially for noncoding regions. Ten gene conversion events between paralogous sequences were inferred. While these events appears to have homogenized regions of otherwise highly differential paralogous sequences, they amplified the differentiation among orthologous sequences. Their effects were larger on coding than on noncoding regions. As a consequence, noncoding sequences grouped by orthologous lineages in phylogenetic trees, whereas coding regions grouped by taxa. Based upon these results, we present a model showing how gene conversion events may also result in the PCR amplification of nonorthologous sequences in different taxa, with obvious complications for phylogenetic inferences, comparative mapping, and population genetic studies. Received: 11 October 2000 / Accepted: 18 September 2001     

Perils of paralogy: using HSP70 genes for inferring organismal phylogenies

Conserved genes have found their way into the mainstream of molecular systematics. Many of these genes are members of multigene families. A difficulty with using single genes of multigene families for phylogenetic inference is that genes from one species may be paralogous to those from another taxon. We focus attention on this problem using heat shock 70 (HSP70) genes. Using polymerase chain reaction techniques with genomic DNA, we isolated and sequenced 123 distinct sequences from 12 species of sharks. Phylogenetic analysis indicated that the sequences cluster with constituitively expressed cytoplasmic heat shock-like genes. Three highly divergent gene clades were sampled. A number of similar sequences were sampled from each species within each distinct gene clade. Comparison of published species trees with an HSP70 gene tree inferred using Bayesian phylogenetic analysis revealed several cases of gene duplication and differential sorting of gene lineages within this group of sharks. Gene tree parsimony based on the objective criteria of duplication and losses showed that previously published hypotheses of species relationships and two novel hypothesis based on Bayesian phylogenetics were concordant with the history of HSP70 gene duplication and loss. By contrast, two published hypotheses based on morphological data were not significantly different from the null hypothesis of a random association between species relatedness and the HSP70 gene tree. These results suggest that gene tree parsimony using data from multigene families can be used for inferring species relationships or testing published alternative hypotheses. More importantly, the results suggest that systematic studies relying on phylogenetic inferences from HSP70 genes may by plagued by unrecognized paralogy of sampled genes. Our results underscore the distinction between gene and species trees and highlight an underappreciated source of discordance between gene trees and organismal phylogeny, i.e., unrecognized paralogy of sampled genes.     

Molecular systematic studies of eubacteria, using sigma70-type sigma factors of group 1 and group 2.

Sigma factors of the sigma70 family were used as a phylogenetic tool to compare evolutionary relationships among eubacteria. Several new sigma factor genes were cloned and sequenced to increase the variety of available sequences. Forty-two group 1 sigma factor sequences of various species were analyzed with the help of a distance matrix method to establish a phylogenetic tree. The tree derived by using sigma factors yielded subdivisions, including low-G+C and high-G+C gram-positive bacteria, cyanobacteria, and the alpha, beta, gamma, and delta subdivisions of proteobacteria, consistent with major bacterial groups found in trees derived from analyses with other molecules. However, some groupings (e.g., the chlamydiae, mycoplasmas, and green sulfur bacteria) are found in different positions than for trees obtained by using other molecular markers. A direct comparison to the most extensively used molecule in systematic studies, small-subunit rRNA, was made by deriving trees from essentially the same species set and using similar phylogenetic methods. Differences and similarities based on the two markers are discussed. Additionally, 31 group 2 sigma factors were analyzed in combination with the group 1 proteins in order to detect functional groupings of these alternative sigma factors. The data suggest that promoters recognized by the major vegetative sigma factors of eubacteria will contain sequence motifs and spacing very similar to those for the sigma70 sigma factors of Escherichia 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.