Nitrogen-regulated hypermutator strain of Synechococcus sp. for use in in vivo artificial evolution.
Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH(4)(+) as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.
Emlyn-Jones
Daniel
D
Molecular Plant Physiology, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
Price
G Dean
GD
Andrews
T John
TJ
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000458:Cyanobacteria / Q000235:genetics / Q000254:growth & development*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D010641:Phenotype; D012293:Rifampin / Q000494:pharmacology; D000198:Spectinomycin / Q000494:pharmacology; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D000198:Spectinomycin; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; D009584:Nitrogen; D012293:Rifampin