Urea sensitization caused by separation of Helicobacter pylori RNA polymerase beta and beta' subunits

BACKGROUND: The beta and beta' subunits of RNA polymerase are fused in all Helicobacters, but separate in most other taxa. Prior studies had shown that this fusion is not essential for viability in culture or in vivo, but had not tested it for potentially important quantitative effects on phenotype. METHODS: The effect of separating rpoB and rpoC sequences on Helicobacter pylori growth was tested in culture and during mouse infection. RESULTS: Derivatives of strains X47 and SS1 carrying this "rpoBCsplit" allele colonized mice less vigorously than their wild-type parents in competition tests. With X47 rpoBCsplit, this reduced vigor was evident in wild-type mice, whereas with SS1 rpoBCsplit it was seen only in cytokine IL-10- and IL-12beta-deficient mice. In culture, the rpoBCsplit allele sensitized each of four strains tested (X47, SS1, 88-3887, and AM1) to urea, a metabolite that is secreted into the gastric mucosa; urea sensitization was more severe in X47 than in SS1 genetic backgrounds. The rpoBCsplit allele also caused poorer growth on Ham's F12 agar, a nutritionally limiting medium, but had little effect on sensitivity to mild acidity. CONCLUSIONS: H. pylori's normal RNA polymerase beta-beta' subunit fusion contributes quantitatively to fitness. We propose that urea, although important to H. pylori in vivo, also be considered inhibitory; and that H. pylori's natural beta-beta' subunit fusion helps it cope with urea exposure.     

Fused and Overlapping rpoB and rpoC Genes in Helicobacters, Campylobacters, and Related Bacteria

The genes coding for the beta (rpoB) and beta' (rpoC) subunits of RNA polymerase are fused in the gastric pathogen Helicobacter pylori but separate in other taxonomic groups. To better understand how the unique fused structure evolved, we determined DNA sequences at and around the rpoB-rpoC junction in 10 gastric and nongastric species of Helicobacter and in members of the related genera Wolinella, Arcobacter, Sulfurospirillum, and Campylobacter. We found the fusion to be specific to Helicobacter and Wolinella genera; rpoB and rpoC overlap in the other genera. The fusion may have arisen by a frameshift mutation at the site of rpoB and rpoC overlap. Loss of good Shine-Dalgarno sequences might then have fixed the fusion in the Helicobacteraceae, even if fusion itself did not confer a selective advantage.     

Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase

Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase. These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences. The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit. The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit. RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene. No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2. Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product. Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E. coli RNA polymerase.     

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.     

Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene.

The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation.     

Molecular analysis of a 21.1-kb fragment of wheat chloroplast DNA bearing RNA polymerase subunit (rpo) genes.

The entire nucleotide sequence of a 21.1-kb fragment of wheat chloroplast (ct) DNA was determined. This fragment carries 18 intact genes and parts of two additional genes, including the three RNA polymerase genes rpoB, rpoC1, and rpoC2. The gene arrangement of this region is conserved in wheat, rice, and maize, but not in non-grass species. Comparison of these 20 genes in wheat, rice, and maize showed that tRNA genes evolved more slowly than protein-coding genes in the chloroplast genome. Intergenic regions evolved much faster than both types of genes. Although the 19 genes of wheat, except for orf42, showed high identity to those of other plants, there were three novel structural features in the wheat rpoC2 gene; a deletion of 81 bp in the middle region, a variable insertion (408 bp), and a nonsense mutation in the 3' terminal region, resulting in truncation of a sequence of ca. 10 amino acids. An intermolecular recombination between the stretches of CTTAT and CTTTT was suggested as the mechanism of the 81-bp deletion in the wheat rpoC2 gene. Evolutionary distance between the chloroplast genomes of wheat and maize was larger than those between wheat and rice and between rice and maize.     

Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli.

The adjacent genes rpoB and rpoC code for the beta and beta' subunits of RNA polymerase in Escherichia coli, and are cotranscribed in the order given. The nearest known genes to rpoB are rplL and rplA,J,K, which code for ribosomal proteins, and which are transcribed in the same direction as the polymerase genes. It has been suggested that rpoBC may be distal elements of a larger operon including these ribosomal genes. To test this possibility we have cloned a segment of DNA, derived by endoR. HindIII digestion from the rpoBC-transducing bacteriophage lambdarifd18, in the replacement vector NMlambda761. The structure of the lambdarpoBC bacteriophages so produced is such that the inserted DNA can be transcribed from lambda promoters, allowing us to confirm that it carries intact rplL, rpoB, and rpoC genes. We have studied these bacteriophages as lysogens in rec+ and rec bacteria, and by infection of UV-irradiated bacterial strains in which lambda promoters are either repressed or active. The results indicate that the cloned DNA contains at most a very weak promoter for the above genes, in contrast to that present in the larger segment of bacterial DNA carried by lambdarifd18. We have in the same way cloned the adjacent bacterial HindIII-fragment of lambdarifd18 DNA, and have found that it displays vigorous autonomous expression of the tufB, rplA, and rplK genes. We conclude that rpoB and C are obligatorily co-transcribed with rplL, from a promoter located outside the DNA segment cloned in lambdarpoBC. We discuss the evidence for the existence of a regulatory site, rpoU, located between rplL and rpoB.     

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Fitness-compensatory mutations in rifampicin-resistant RNA polymerase

Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.