Identiﬁcation and Characterization of a Locus Which Regulates Multiple Functions in Pseudomonas tolaasii , the Cause of Brown Blotch Disease of Agaricus bisporus

Pseudomonas tolaasii , the causal agent of brown blotch disease of Agaricus bisporus , spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans . Subcloning and saturation mutagenesis analysis with Tn 5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn 5lacZ -mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN :: lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii. qualitative analysis 57). The spontaneous phenotypic vari- ant 1116R differed from the wild type in its inability to synthesize tolaasin or to hydrolyze casein as well as its ability to synthesize larger amounts of ﬂuorescent pigments, to swim faster, and to grow more rapidly than 1116S. In addition, 1116R was more sensitive to both the membrane-acting antibiotic polymyxin and the intracellular antibiotic nalidixic acid. Analysis for the presence of plasmid DNA revealed that no indigenous plasmids were present in either 1116R or 1116S strains of P. tolaasii . Reversion of 1116R to 1116S at a frequency of approximately 1 in 10 6 cells was observed when an actively growing single cell isolate of 1116R was analyzed. Cloning of the pheN locus involved in phenotypic variation of P. tolaasii. A cosmid bank of 1116S chromosomal DNA in the broad-host-range vector pLAFR3 was transferred en masse by conjugation to 1116R. A total of 3,000 transconjugants were screened for colonies showing a wild-type colony morphology, tolaasin production, and ability to hydrolyze casein. Cosmid clones isolated from these transconjugants were introduced individually in 1116R to test for restoration of the wild-type phenotype. Six out of the resulting 10 clones isolated were capable of reverting virtually 100% of the 1116R population to Phe (cid:49) . An identical restriction banding pattern for enzymes Eco RI, Hin dIII, and Pst I in all six clones (results not shown) demonstrated the presence of a cosmid with the same 29.9-kb insert in each case, which was designated pSISG29. to Phe quantitatively biochemical characteristics of merodiploids with those of the 1116S and 1116R forms. revealed that the 1116R(pSISG29)