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.     

Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168.

The nucleotide sequence of a 2.14 kb fragment of Bacillus subtilis DNA containing the citG gene encoding fumarase was determined using the dideoxy chain termination method. The citG coding region of 1392 base pairs (464 codons) was identified, and the deduced Mr (50425) is in good agreement with that of the protein identified from expression in Escherichia coli maxicells. There is no sequence homology between the B. subtilis and E. coli fumarases. Overlapping potential promoter sequences have been identified for sigma 28, sigma 37 and sigma 55 RNA polymerase holoenzymes. The DNA fragment also contains the proximal part of the gerA locus, responsible for L-alanine-sensitive spore germination.     

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis . The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal σ factors of a number of divergent prokaryotes. It is larger than most σ factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285. Furthermore, B. pertussis rpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence-associated fha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.     

Replication mutations differentially enhance RecA-dependent and RecA-independent recombination between tandem repeats in Bacillus subtilis

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.