Abstract
The ability to introduce random mutations into genes cloned in bacterial vectors is particularly useful for bioengineering and molecular biological studies. The increasing importance of hypermutable bacteria for these purposes is emphasized by the commercial availability of the E. coli strain XL1-Red (Stratagene), which contains mutS, mutT and mutD mutations. Defects in mutS confer a hypermutable phenotype because this gene is part of the methyl directed mismatch repair (MMR) system, a post-replicative DNA repair pathway that identifies and corrects mismatched DNA duplexes (LeClerc et al., 1996). MutT mutator strains are unable to hydrolyse 8-oxodGTP and display an increase in transversions (Fowlera & Schaaper, 1997). Defects in mutD also contribute to the hypermutability of strain XL1-Red because the frequency of transitions and transversions is enhanced by the loss of the DNA polymerase proofreading function (Selifonova et al., 2001).
During preliminary experiments designed to select mutant SHV-1 β-lactamases with altered substrate specificities, we constructed a recombinant, designated pJMBBle+, which contained the gene for SHV-1 (Mercier & Levesque, 1990) in the common cloning vector pCR-Blunt (Invitrogen). Introduction of pJMBBle+ into E. coli hypermutators in regular use in our laboratory (Miller et al., 2002) and strain XL1-Red appeared to suppress mutation rates for β-lactam resistance. Plasmid pCR-Blunt and several other commercially available cloning and expression vectors (Invitrogen) contain the Zeocin-resistance gene ShBle. Expression of ShBle in prokaryotic and eukaryotic hosts confers resistance to the broad-spectrum antibiotic Zeocin. To investigate a possible role for the ShBle gene product in the suppression of mutation frequencies in this system, we excised the ShBle gene from pJMBBle+ using the restriction enzyme PmlI (New England Biolabs), resulting in the plasmid pJMBBle-. We then compared the effects of pJMBBle+ and pJMBBle- on endogenous antibiotic resistance mutation frequencies in a number of E. coli mutator strains, including XL1-Red (Table 1).