Genetic and Transcriptional Organization of the Region Encoding the β Subunit of Bacillus subtilis RNA Polymerase

Abstract

The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a lambda gt11 expression library using an antibody probe. Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region. Including the rif region, four colinear blocks of sequence similarity were shared between the B. subtilis and E. coli beta subunits. In E. coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B. subtilis protein. The B. subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta'). This organization differs from the corresponding region in E. coli by the inclusion of orf23. Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3'-coding region of orf23, 250 nucleotides upstream from the beta initiation codon. Thus, the B. subtilis rpoB region differs from its E. coli counterpart in both genetic and transcriptional organization.

Mycobacterium kyorinense sp. nov., a novel, slow-growing species, related to Mycobacterium celatum, isolated from human clinical specimens

“…DNA was prepared after mechanical disruption of bacterial cells and subjected to sequence analysis of the 16S rRNA, hsp65, rpoB and sodA genes using methods as described by Kirschner et al (1993), Telenti et al (1993), Boor et al (1995) and Domenech et al (1997) with some modifications. Phylogenetic trees with bootstrap values were generated using the CLUSTAL W program (www.clustal.org) and displayed using TREEVIEW as described by Li et al (2004).…”

Mutations in the β subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG

“…PCRScript clones were transformed into wild-type (Rif s ) B. subtilis 168 with selection for Rif r by homologous recombination of clusters I-III in a double cross-over event (Boor et al, 1995). PCRScript clones were concluded to contain a Rif r mutation if they transformed B. subtilis 168 to Rif r at a higher efficiency ( 10-fold) than the same region of rpoB derived from a Rif s strain in at least two independent transformation experiments (Boor et al, 1995). PCRScript vectors containing rpoB fragments conferring a Rif r phenotype were sequenced using an ABI automated sequencer.…”

“…Additional mutant selections performed after mutagenesis with EMS or NTG did not yield a greater variety of Rif r mutants (seven mutants mapped and sequenced). The mutation H482Y has previously been isolated as rfm2103 in B. subtilis Marburg (Boor et al, 1995). Mutations identical to Q469R, H482Y and Q469K have also been reported in E. coli (Jin & Gross, 1988 ;Severinov et al, 1993 ;Yang & Polisky, 1999).…”

Analysis of Core Housekeeping and Virulence Genes Reveals Cryptic Lineages ofClostridium perfringensThat Are Associated With Distinct Disease Presentations

“…found within the rplL FR in Proteobacteria (e.g., Escherichia coli; Morgan et al 1984), Spirochaetes (e.g., Borrelia burgdorferi; Alekshun et al 1997), and Firmicutes [e.g., Bacillus subtilis (Boor et al 1995) and Staphylococcus aureus (Aboshkiwa et al 1995)]. While we do not have experimental evidence for the presence of regulatory elements in this region of the C. perfringens genome, it is likely because the operon in which rplL is found is under intense purifying selection and is one of the most highly conserved operons in bacteria (Dabbs 1984).…”