Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach.

We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC-binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.

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