A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

Abstract

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toWC and dnaG, at 66 min. The properties of homologous components of the transcription and translation apparatuses often vary between strains of enteric bacteria. Such natural differences provide a convenient tool for genetic mapping of these components when mutants are not available. In order to make use of such differences, investigators individually transfer small regions of the chromosome from one bacterial strain to another strain. The recipients are then screened for the desired phenotype, by using an appropriate assay. With this technique, it has been possible to map some of the genes coding for ribosomal proteins (1) as well as the gene coding for transcription termination factor rho (2). Until now, however, no genetic mapping has been reported for the RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) sigma subunit, which determines the selectivity of transcription in vitro (3). We have observed electrophoretic differences between the RNA polymerase sigma subunits of Escherichia coli strain K-12 (K), E. coli strain C, and Salmonella typhimurium, and have used these differences to map an E. coli K gene which causes synthesis of the sigma subunit characteristic of E. coli strain K.MATERIALS AND METHODS Chemicals and Buffers. Buffers and DNA-cellulose were prepared as described by Burgess and Jendrisak (4 Bacterial Strains. The bacterial strains used are described in Table 1. New merodiploid strains were constructed by transferring F' plasmids from E. coli K merodiploid strains into various Salmonella typhimurium and E. coli C strains, each containing nutritional mutations such that the presence of the F' plasmid would allow growth on minimal glucose medium. Transfer was accomplished by either of two methods. Method 1: a minimal glucose plate was spread with 0.1 ml (107-108 colony-forming units) of an overnight culture of the donor strain and 0.05 ml of the recipient strain, followed by incubation at 370 for 48 hr (13). Method 2: 0.5 ml samples of each overnight culture were inoculated into flasks containing 10 ml of minimal glucose medium supplemented with the nutritional requirements of each strain. These cultures were shaken at 370 until the optical density of the suspension was doubled. One milliliter of the recipient strain, 2 ml of the donor strain and 3 ml of LB broth were then added to a 250 ml Erlenmeyer flask and shaken very slowly at 370 for 60-90 min. The mating mixtures were centrifuged, concentrated to 10% of the initial volume, and 0.2 ml aliquots were spread on minimal glucose plates, followed by incubation at 370 for 48 hr. C...

Klebsiella aerogenes catabolite gene activator protein and the gene encoding it (crp)

“…Similarly, P-galactosidase from K. aerogenes differs greatly in primary sequence from that of E. coli (9), and the K. aerogenes P-galactosidase is far more thermolabile under standard assay conditions than the E. coli enzyme (4). Even the sigma subunits of RNA polymerase from E. coli K-12, E. coli C, and S. typhimurium migrate differently on sodium dodecyl sulfate-polyacrylamide gels (19,21).…”

A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase

“…One way to regulate global gene expression is to modulate the interaction of different a factors with core RNA polymerase in response to physiological and environmental changes (19,26,37). a70, the major a factor in E. coli, is encoded by rpoD (12,18,31) and is essential for cell growth (17,25,35). The rpoD285 mutation and the genetically identical rpoD800 mutation confer a temperature-sensitive (Ts) growth phenotype (17,20,25,30,35) because this mutant sigma factor is unstable and rapidly degraded at high temperature (12, 13).…”

“…a70, the major a factor in E. coli, is encoded by rpoD (12,18,31) and is essential for cell growth (17,25,35). The rpoD285 mutation and the genetically identical rpoD800 mutation confer a temperature-sensitive (Ts) growth phenotype (17,20,25,30,35) because this mutant sigma factor is unstable and rapidly degraded at high temperature (12,13).…”

Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome

“…This mapping method depends upon the direct quantitation of RRF after separation of total cell proteins by two-dimensional gel electrophoresis (29). Although the method itself has been used by several investigators, the quantitation has been performed solely by immunological means (12,28). In mapping the tRNA genes, Ikemura and Ozeki (19) used two-dimensional gel electrophoresis for separation and subsequent quantitation of each tRNA species.…”