EXPRESSION OF THE AGGA LOCUS OF PSEUDOMONAS-PUTIDA IN-VITRO AND IN PLANTA AS DETECTED BY THE REPORTER GENE, XYLE

In vitro agglutinability by Pseudomonas putida, isolate Corvallis, wit h a p]ant root surface agglutinin is correlated with rapid adhesion of cells of the fluorescent pseudomonad to bean (Phaseolus vulgaris) roo t surfaces. Agglutinability in P. putida cells is regulated by nutrien t status as well as growth phase. Cells grown in three different nutri ent complex media are agglutinable at early and mid-late logarithmic p hase but become nonagglutinable at stationary phase. Cells grown in a minimal medium are weakly agglutinable, but the addition of lysine, as partic acid, or histidine increases agglutinability. Cells in the same minimal medium supplemented with bean root surface components prow in a highly agglutinated state. Previous data indicate both agglutinatio n and rapid adhesion to roots by P. putida Corvallis involves the aggA locus, which contains two putative open reading frames (ORF), ORFAGG1 and ORFAGG2, on complementary strands. Sequence and deletion analyses suggest ORFAGG1 is the most probable ORF responsible for agglutinatio n and adhesion. Chimeric fusion of an Escherichia coli lac promoter wi th ORFAGG1, but not with ORFAGG2, complemented agglutinability of an a ggA::Tn5 P. putida Agg(-) mutant, providing further evidence that ORFA GG1, not ORFAGG2, is responsible for agglutination. Heterologous expre ssion of ORFAGG1 yields a 50-kDa precursor and a 48-kDa mature peripla smic protein. Fusions of ORFAGG1 and ORFAGG2 to the reporter gene, xyl E, and detection of the reporter enzyme, catechol-2,3-oxygenase reveal an active promoter in the 5' noncoding region of ORFAGG1. The ORFAGG1 promoter is active during growth of the cells in liquid culture and i s regulated by growth medium. Greatest activity of the catechol-2,3-ox ygenase is observed in stationary phase when the cells are nonagglutin able. Expression of the ORFAGG1 promoter is detected in P. putida cell s extracted from the root surface of bean at 48 and 72 hr after inocul ation.