Site-directed Mutagenesis of the Metal-reducing Bacterium S. oneidensis MR-1: A Novel Strategy for Genetic Engineering in Recalcitrant Microorganisms
Brentwood School, Los Angeles, CA
Shewanella oneidensis MR-1 is widely employed as a model organism for the study of subsurface bioremediation and electricity production in microbial fuel cells. However, its usefulness as a model organism is limited by recalcitrance to classical techniques of genetic engineering. My objective was to implement a strategy for site-directed mutagenesis of the MR-1 chromosome independent of exogenous recombinase functions; to assess and optimize this utility for the construction of point substitutions and large deletions by examining strand-, site- and sequence-specific effects. Competent cells were electroporated with DNA oligonucleotides (oligos) that contained the desired sequence alterations (conferring drug resistance) flanked by homology to the target loci. Recombination frequencies were determined by plating on selective media while controlling for basal mutation rate, with subsequent restriction-fragment analysis to confirm presence of the intended genotype. Point substitutions were achieved at frequencies of 10^-8 to 10^-7, with a lagging-strand bias (p < 0.05). Oligos with 1 silent base change designed to evade mismatch repair (MMR) enhanced the efficiency by ~10-fold (p < 0.05), but oligos with 2 MMR-refractory modifications decreased the efficiency (p < 0.1). Although the drug resistance assay was not sensitive enough to detect oligo-mediated knockouts, PCR amplification revealed that a minority of isolates contained the intended deletion. These findings greatly expand the molecular toolkit for rational manipulation of Shewanella spp. and have broad applicability to other recalcitrant microbes. Ongoing work involves use of long ssDNA substrates to mediate insertion of marker cassettes, obviating the need for mutations that confer selectable traits in themselves.