Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1.
Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a DeltaumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.
Koch
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WH
Molecular Biology Branch, Food and Drug Administration, 20204, Washington, DC, USA.
Fernández de Henestrosa
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AR
Woodgate
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R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005784:Gene Amplification; D005798:Genes, Bacterial; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D010957:Plasmids / Q000235:genetics*; D011815:R Factors / Q000235:genetics; D013014:SOS Response, Genetics; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D008741:Methyl Methanesulfonate; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C065793:ImpC protein, Bacteria; C068606:mucAB protein, E coli; C078202:UmuC protein, E coli