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, with a p]ant root surface agglutinin is correlated with rapid adhesion of cells of the fluorescent pseudomonad to bean (Phaseolus vulgaris) root surfaces. Agglutinability in P. putida cells is regulated by nutrient status as well as growth phase. Cells grown in three different nutrient complex media are agglutinable at early and mid-late logarithmic phase but become nonagglutinable at stationary phase. Cells grown in a minimal medium are weakly agglutinable, but the addition of lysine, aspartic 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 agglutination 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 agglutination and adhesion. Chimeric fusion of an Escherichia coli lac promoter with ORFAGG1, but not with ORFAGG2, complemented agglutinability of an aggA::Tn5 P. putida Agg(-) mutant, providing further evidence that ORFAGG1, not ORFAGG2, is responsible for agglutination. Heterologous expression of ORFAGG1 yields a 50-kDa precursor and a 48-kDa mature periplasmic protein. Fusions of ORFAGG1 and ORFAGG2 to the reporter gene, xylE, 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 is regulated by growth medium. Greatest activity of the catechol-2,3-oxygenase is observed in stationary phase when the cells are nonagglutinable. Expression of the ORFAGG1 promoter is detected in P. putida cells extracted from the root surface of bean at 48 and 72 hr after inoculation.