The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon.

The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.     

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.     

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.     

An RpoH-like heat shock sigma factor is involved in stress response and virulence in Brucella melitensis 16M

B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD, rpoH1, rpoH2, rpoE1, rpoE2, and rpoN. We mutated all these genes except rpoD. Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (DeltarpoH2) is impaired for growth at 21 and 42 degrees C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the DeltarpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (DeltarpoH1, DeltarpoE1, and DeltarpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis, the type IV secretion system and the flagellum.     

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.