Antimicrobial Research Centre and Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Julieanne Bostock
(j.m.bostock{at}leeds.ac.uk)
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 -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
-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
).
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The mechanism of action of Zeocin is unknown, but may be similar to that of the structurally related group of bleomycin/phleomycin antibiotics (Berdy, 1980). The bleomycinFe(II) complex, in conjunction with reducing agent and oxygen, causes nucleotide-specific DNA cleavage. The ShBle gene, isolated from Streptoalloteichus hindustanus (Gatignol et al., 1988
), encodes a protein that binds stoichiometrically to bleomycin, inhibiting its DNA strand cleavage activity (Dumas et al., 1994
).
In transposon Tn5, the bleomycin-resistance phenotype encoded by the ble gene is associated with improved host fitness, even in the absence of the drug (Blot et al., 1991). Improved fitness may be mediated by enhancement of the DNA repair system, although the exact role of ble has not been determined. Nevertheless, bleomycin resistance mediated by ble appears to be independent of its ability to confer a fitness advantage on the host bacterium (Kumagai et al., 1999
). The ShBle and Ble proteins share 25 % amino acid identity. Both proteins are acidic and exhibit low isoelectric points and low molecular masses. These features suggest that their structural genes are derived from a common ancestor (Kumagai et al., 1999
). Therefore, the ShBle protein may enhance DNA repair via the same mechanism as Ble. This enhancement could be sufficient to compensate for the defective mismatch repair and proofreading activities in the hypermutators that lack one or more of these error-correcting systems. However, in cells with a fully proficient set of DNA repair genes no further enhancement of repair is conferred by the ShBle protein.
Mutation analysis is an invaluable tool to investigate relationships between genotype and phenotype. The introduction of random mutations into cloned genes by hypermutable hosts is an easy and efficient approach to investigate this relationship. However, we would advise close monitoring of mutation frequencies to avoid inadvertent suppression of mutation by markers that may be present in cloning vectors to assist selection of recombinants. This will also apply to in vivo mutagenesis with alkylating agents since the Ble protein interferes with the mutagenic potential of such compounds (Blot et al., 1991).
REFERENCES
Berdy, J. (1980). Bleomycin-type antibiotics. In Amino Acid and Peptide Antibiotics. Handbook of Antibiotic Compounds, IV (1). Edited by J. Berdy. Boca Raton, FL: CRC Press.
Blot, M., Meyer, J. & Arber, W. (1991). Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12. Proc Natl Acad Sci U S A 88, 91129116.[Abstract]
Dumas, P., Bergdoll, M., Cagnon, C. & Masson, J. M. (1994). Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering. EMBO J 13, 24382492.
Fowlera, R. G. & Schaaper, R. M. (1997). The role of the mutT gene of Escherichia coli in maintaining replication fidelity. FEMS Microbiol Rev 21, 4354.[CrossRef][Medline]
Gatignol, A., Durand, H. & Tiraby, G. (1988). Bleomycin resistance conferred by a drug-binding protein. FEBS Lett 230, 171175.[CrossRef][Medline]
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Kumagai, T., Nakano, T., Maruyama, M., Mochizuki, H. & Sugiyama, M. (1999). Characterization of the bleomycin resistance determinant encoded on the transposon Tn5. FEBS Lett 442, 3438.[CrossRef][Medline]
LeClerc, J. E., Li, B., Payne, W. L. & Cebula, T. A. (1996). High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274, 12081211.
Mercier, J. & Levesque, R. C. (1990). Cloning of SHV-2, OHIO-1, and OXA-6 -lactamases and cloning and sequencing of SHV-1
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Miller, K., O'Neill, A. J. & Chopra, I. (2002). Response of Escherichia coli hypermutators to selection pressure with antimicrobial agents from different classes. J Antimicrob Chemother 49, 925934.
Selifonova, O., Valle, F. & Schellenberger, V. (2001). Rapid evolution of novel traits in microorganisms. Appl Environ Microbiol 67, 36453649.
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