The Secretion Of Lecithinase Was Via Type Ⅱ Secretion Pathway In Pseudomonas Alcaligenes S2

The purpose of our research is to isolate the mutants affecting solubilization of organic phosphate and to study the genes involved in the solubilization of organic phosphate of Pseudomonas alcaligenes S2. S2, a phosphatidylcholine (or lecithin) solubilizing bacterium, was isolated from the rice rhizosphere in rural areas of Beijing, China. A transposon Tn5-1063 mutagenesis library containing over 5000 clones of P. alcaligenes S2 was constructed and four mutants were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of P. alcaligenes S2 involved in solubilization of phosphatidylcholine, the EcoR I fragments of the chromosomes from four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. DNA sequence analysis showed that the Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decrease solubilizing ability of phosphatidylcholine, were found to be located in the xcpS, xcpX and xcpW respectively, encoding the proteins XcpS, XcpX and XcpW of the type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize phosphatidylcholine. In the mutant M20 showing increased phosphatidylcholine-hydrolyzing activity, the interrupted gene was chpA. Four mutants affecting solubilization of phosphatidylcholine were obtained in this study. The secretion of phosphatidylcholine-hydrolyzing enzyme of Pseudomonas alcaligenes was via the type II secretion pathway.