Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, p...Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G+C content determination and DNA-DNA hybridizations analy-sis, strain S2 was identified as Pseudomonas alcaligenes. P. alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin 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 lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of leci-thin-hydrolyzing enzyme of P. alcaligenes was via type II secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitch-ing motility of the bacterial cells.展开更多
NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interac...NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7 genomic libraries by using the yeast two-hybrid sys- tem with NifA as bait. We are interested in clone S35, one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35 gene is 2211-bp long and encodes a protein of 736 aa with a predicted molecular weight of about 78.4 kD. The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Struc- tural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.展开更多
文摘Strain S2 is a lecithin (or phosphatidylcholine)- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G+C content determination and DNA-DNA hybridizations analy-sis, strain S2 was identified as Pseudomonas alcaligenes. P. alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin 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 lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type II secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of leci-thin-hydrolyzing enzyme of P. alcaligenes was via type II secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitch-ing motility of the bacterial cells.
基金This work was supported by the National Natural Science Foundation of China （Grant No. 30470028）.
文摘NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7 genomic libraries by using the yeast two-hybrid sys- tem with NifA as bait. We are interested in clone S35, one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35 gene is 2211-bp long and encodes a protein of 736 aa with a predicted molecular weight of about 78.4 kD. The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Struc- tural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.
