RESUMEN
When subcloned into low-copy-number expression vectors, rumAB, encoding polVR391 (RumA'2 B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polVR391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polVR391 . Indeed, we recently discovered that SetRR391 , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroSR391 , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetRR391 and CroSR391 , with the greatest reduction of RumA protein levels observed in the absence of SetRR391 and the presence of CroSR391 . Under these conditions, CroSR391 completely abolishes the high levels of mutagenesis promoted by polVR391 expressed from low-copy-number plasmids. Furthermore, deletion of croSR391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroSR391 plays a major role in suppressing polVR391 mutagenesis in vivo. Inactivating mutations in CroSR391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391.
Asunto(s)
Bacteriófago lambda/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virología , Metiltransferasas/metabolismo , Mutagénesis , Proteínas Represoras/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Respuesta SOS en Genética , Eliminación de SecuenciaRESUMEN
The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.