Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA p olymerases suggest that they can be broadly divided into four sub-groups co mprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The Umu C-like sub-family is best characterized at the genetic level and sequence a nalysis of eleven umu orthologs, residing on bacterial chromosomes or on se lf-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conser vation. Some of these orthologs are apparently inactive in situ, but may pr omote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an as say that would identify umuC-like genes in situ in the absence of a functio nal assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were desig ned and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1. Interestingly, DNA sequence analysis of an similar to7 kb region of R394 id entified two LexA-regulated genes immediately downstream of mucAB((R394)) t hat 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 r estore mutagenesis functions to a Delta umuDC E. coli strain, Rts-1 surpris ingly promoted significant levels of MMS-induced SOS mutagenesis, raising t he possibility that Rts-1 encodes another, yet unidentified, umu-like homol og. (C) 2000 Elsevier Science B.V. All rights reserved.