Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Characterization of cycA mutants of Escherichia coli: An assay for measuring in vivo mutation rates
Introduction
The genomic mutation rate is a fundamental evolutionary parameter of any population. Cells maintain a balance between faithful replication and mutation-generating mechanisms. While most mutations are deleterious, genetic diversity on which selection can work is a necessity for long-term survival in a changing environment. Genetic adaptation was shown to be an uneven process. Certain environmental stresses can result in increased mutation rates creating higher genetic diversity [1], [2], although the long-term selective advantage of such temporary mutagenesis in some genetic systems has been brought into question [3].
Mutations in bacteria occur typically at a rate of <10−6/gene/replication [4]. Analysis of such rare events usually requires the use of a screen, preferably a positive selection system which filters out the mutants from a much larger population. Ideally, the mutation detection system selects mutations which (i) can be of any type, (ii) are neutral in respect of cell growth, (iii) do not require a specific genetic background, (iv) have a simple phenotype–genotype relationship and (v) can be easily and reproducibly counted.
The work horse of molecular biology, Escherichia coli, is a prominent subject of mutation research. A wealth of information on mutations occurring under various conditions was derived from studies performed with this bacterium, including recent observations on the increase of starvation-induced point mutation rates [5], or detection of beneficial insertion element-mediated rearrangements in long-term-cultured cells [6].
A great number of mutation detection systems have been used in E. coli for mutagenesis studies [7], however, the use of them is frequently limited by various factors. Narrow range of mutation types detected, interference with cell metabolism/growth, need for specific genetic background, multiple mutation target genes, multicopy phage- or plasmid-coded components are the causes of limitations associated with the mutation selection systems. In the course of our work studying insertion element (IS)-mediated mutagenesis, we developed an assay, based on the selection of cycA mutants, which proved to be particularly useful for the analysis of mutation rates and spectra. We provide a characterization of this system here.
The cycA gene of E. coli codes for a permease locating in the outer membrane and transporting d-alanine, d-serine, and glycine into the cell [8], [9], [10], [11]. d-Cycloserine, an antibiotic interfering with cell wall synthesis [12], is also transported by CycA [13], [14]. Mutations in CycA render the cell resistant to d-cycloserine. Since d-cycloserine has multiple targets (d-alanyl-d-alanine ligases A and B, and alanine racemase) [15], [16], [17], [18], the simultaneous modification of which can practically be excluded, we hypothesized that resistance under the conditions of the assay is exclusively due to mutations of the transporter, CycA. We show here that cycA mutants resistant to d-cycloserine can carry any type of mutations (point mutations, insertions, deletions), and these mutations tend to be neutral for growth in minimal medium. Results obtained by the test are reproducible, and the assay is easy to perform. It is based on a simple phenotype–genotype relationship, there is no requirement for a specific genetic background, and mutations are detected in a single-copy, chromosomally located gene.
Section snippets
Strains, plasmids and media
Mutation rate experiments were done using K-12 MG1655 [19]. MDS42, a deletional derivative of MG1655, will be described in detail elsewhere. Plasmids were prepared from DH5α host. To clone cycA of MG1655, a 1877-bp genomic region encompassing cycA was amplified using flanking primers cycA1R (5′-cggaattcatggtagatcaggtaaaagt) and cycA2R (5′-accccaagcttgcgccatccagcatgata). The amplified fragment was cloned into the EcoRI–HindIII sites of pBADαβγ [20], downstream of the inducible promoter.
Selection of d-cycloserine resistant mutants
For selection of d-cycloserine mutants, the inhibitory effect of the antibiotic was studied in the 0.01–0.06 mM range on glucose/minimal plates. Earlier reports defined 0.01 mM as the minimal concentration of d-cycloserine needed to completely inhibit growth of E. coli K-12 on solid medium [9]. We found that 0.02 mM inhibited growth on agar plates. However, for complete elimination of the background of slow-growing non-resistant cells, use of 0.04 mM was necessary. For routine assays, cells were
Discussion
A short sampling of the frequently used mutation selection systems in E. coli [7] reveals that many of them provide only partial information on the mutational events in the cell. Variants of the widely used assays utilizing the lac operon provide only a screen, limiting the quantitative analysis. Among the positive selection systems, the frequently used rifampicin test detects only base substitutions. Moreover, we show here that these mutations can affect the growth rate. The test based on the
Acknowledgements
We are grateful for the expert technical assistance provided by Ágnes Szalkanovics. This work was supported by grant OTKA T043260 to G.P.
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