PHENOTYPE CONVERSION IN PSEUDOMONAS-SOLANACEARUM DUE TO SPONTANEOUS INACTIVATION OF PHCA, A PUTATIVE LYSR TRANSCRIPTIONAL REGULATOR

Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinat ed change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence. Alth ough PC is normally spontaneous, it is mimicked by transposon inactiva tion of the phcA locus (S. M. Brumbley and T. P. Denny, J. Bacteriol. 172:5677-5685, 1990). The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should enc ode a polypeptide of 38.6 kDa. The PhcA protein produced in Escherichi a coli by using a T7 RNA polymerase expression system was of the predi cted size. The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, esp ecially in the amino terminus, where a putative helix-turn-helix DNA-b inding motif was identified. An analogous allele (phcA1) was cloned fr om the spontaneous PC mutant strain AW1-PC and found to be nonfunction al in complementation studies. When phcA1 was expressed in E. coli, th e PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA. Sequence analys is of phcA1 and chimeric constructs of phcA and phcA1 confirmed that P hcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA. Southern blot analysis of 10 additional ind ependently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA. These results su ggest that phcA encodes a DNA-binding protein that regulates the trans cription of one or more of the genes involved in P. solanacearum virul ence and that spontaneous PC can be attributed to one of several diffe rent insertions within this locus.