CHARACTERIZATION OF GENES REQUIRED FOR PILUS EXPRESSION IN PSEUDOMONAS-SYRINGAE PATHOVAR PHASEOLICOLA

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv, phaseolicola were generated by Tn5 transposon mutagenesis. A P. syrin gae pv. phaseolicola LR700 cosmid library was screened with Tn5-contai ning EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb seq uenced region spanning the Tn5 insertion site contained four open read ing frames. The transposon-inactivated gene, designated pilP, is 525 b p long, potentially encoding a 19.1-kDa protein precursor that contain s a typical membrane lipoprotein lender sequence, Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expressi on of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa , in agreement with the sizes of the open reading frames, The three ge nes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature f orm was shown to be inhibited by globomycin, a specific inhibitor of s ignal peptidase II. The gene region identified shows a high degree of homolog to a gene region reported to be required for Pseudomonas aerug inosa type IV pilus production.