Construction and Characterization of Pseudomonas syringae recA Mutant Strains. David K. Willis. USDA/ARS Plant Disease Resistance Research Unit, Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706. Estelle M. Hrabak(2), Steven E. Lindow(3), and Nickolas J. Panopoulos(3). (1) USDA/ARS Plant Disease Resistance Research Unit and (2) Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706; and (3) Department of Plant Pathology, University of California, Berkeley, California 94720 U.S.A.. MPMI 1:80-86. Accepted 20 January 1988. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1988.

We have constructed recA mutant derivatives of both pathogenic and nonpathogenic strains of Pseudomonas syringae using two methods of site-directed mutagenesis. Two plasmids were constructed, one nonreplicative (pKW11) and one replicative (pEMH1) within P. syringae, each containing a 19.2-kb EcoRI fragment carrying the P. syringae recA4::Tn5 mutation. Using pKW11, the nonpathogenic strain Cit7 (source of the P. syringae recA gene) was mutagenized by the apparent creation and resolution of a cis-merodiploid intermediate. The resultant strain, Cit7 (recA4::Tn5), exhibited sensitivity to ultraviolet irradiation characteristic of an Escherichia coli recA mutant. In order to test the effect of a recA genetic background on pathogenicity and in planta growth, we used pEMH1 to mutagenize, by the transplacement method, the heterologous P. syringae pv. syringae isolate B728a, a causal agent of bacterial brown spot disease of bean (Phaseolus vulgaris). The resultant mutant, B728a (recA4::Tn5), exhibited a degree of UV-sensitivity similar to Cit7 (recA4::Tn5) by quantitative assay. Strain B728a (recA4::Tn5) was not altered in its ability to grow in culture or in planta or in its ability to cause disease in the leaves or pods of bean. The ability to mutagenize heterologous strains of P. syringae by gene replacement without any apparent effect on in planta growth and pathogenicity indicates that a recA deficient background should be generally useful in the genetic analysis of phytopathogenic pseudomonads.