SPONTANEOUS DUPLICATION OF A 661 BP ELEMENT WITHIN A 2-COMPONENT SENSOR REGULATOR GENE CAUSES PHENOTYPIC SWITCHING IN COLONIES OF PSEUDOMONAS-TOLAASII, CAUSE OF BROWN BLOTCH DISEASE OF MUSHROOMS

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a ph enotypic switch from the smooth, pathogenic form (designated 1116S) to the rough nonpathogenic form (designated 1116R), This phenotypic swit ch can also be induced by mutation of the pheN master regulatory locus , which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins, Southern blot analysis of genomic DNA f rom 1116S and 1116R probed with a 3.4 kb Xhol-BamHI fragment containin g the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R, In order to characterize the genetic basi s of this variation, the pheN locus (designated pheN') was cloned from 1116R and its nucleotide sequence determined, A 661 bp duplication wa s identified within pheN' introducing a frameshift mutation in the pre dicted pheN open reading frame (ORF), A resulting predicted ORF of phe N' designated ORF2 encodes a polypeptide of 706 amino acid residues, w ith a predicted molecular weight of 77 kDa, and which lacks part of th e PheN sensor domain, Southern blot analysis of genomic DNA using a pr obe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form, Polymerase chain reactio n (PCR) analysis of 25 independently isolated 1116R sectors using prim ers flanking the duplication site in pheN confirmed the presence of th e duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants f rom 1116R to 1116S, Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form . The presence of a truncated PheN protein in 1116R was verified by We stern blot analysis of total cell protein using a LemA antiserum, whic h revealed the presence of 99 kDa and 77 kDa cross-reactive bands in 1 116S and 1116R respectively. It is concluded that the spontaneous colo ny-sectoring event that results in the 1116R phenotypic variant form o f P. tolaasii arises owing to a 661 bp DNA duplication within the 5' e nd of the pheN gene, which results in loss of the periplasmic sensor d omain of PheN and elimination of normal PheN function.