redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD.

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.     

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.     

Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.     

Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin

A fragment of DNA carrying the hitherto unisolated members of the cluster of genes (red) for biosynthesis of the red-pigmented antibiotic undecylprodigiosin of Streptomyces coelicolor A3(2) was isolated. This was done by cloning random fragments of S. coelicolor DNA into the closely related Streptomyces lividans 66 and recovering a clone that caused overproduction of undecylprodigiosin. The effect was probably due to the presence of the cloned redD gene, which functions as a positive regulator of the expression of the red cluster, activating the normally poorly expressed red genes of S. lividans. Two fragments from either end of the red cluster were cloned adjacent to each other on a low-copy-number Streptomyces vector. Double crossing-over occurring between these plasmid-borne sequences and the chromosomal copy of the same DNA in S. coelicolor led to isolation of the entire red cluster as a single cloned fragment. Isolation of antibiotic biosynthetic genes by the effects of an activator in a self-cloning experiment, and in vivo reconstitution of a large cluster of genes by homologous recombination, may turn out to be usefully generalizable procedures.     

Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli

Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E. coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes or stable RNA. We have found that Rifr mutations affect both lambda N-mediated antitermination and the cellular antitermination system involved in synthesis of stable RNA. Because NusA is involved in antitermination and termination, we also investigated the interaction of NusA and RNA polymerase by determining whether Rifr mutations alter NusA-dependent termination or antitermination in cells with defective nusA alleles. We have shown that Rifr mutations can either enhance or suppress the phenotypes of defective nusA alleles. Most Rifr mutations alter the temperature range over which the nusA1 allele supports lambda N-mediated antitermination. In addition, a number of Rifr alleles restore termination to the nusA10(Cs) and the nusA11(Ts) mutants defective in this process. Our results indicate that the region of the rpoB gene defined by the Rifr mutations is involved in the antitermination process and affects the activity of the NusA protein directly or indirectly.     

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.     

Genes that protect against the host-killing activity of the E3 protein of Bacillus subtilis bacteriophage SPO1.

A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.