| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 93 === Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium causing black rot in cruciferous plants. The virulence of Xcc depends on a number of pathogenic genes and virulent factors including the exopolysaccharide and extracellular enzymes such as proteases, endoglucannases, and pectinases. It is known that extracellular enzymes in various Gram-negative bacteria are secreted by type II secretion pathway using PilD as the signal pepetidase for N-terminus processing.
A pilD mutant, MC1220 (pilD::pSMP104), has previously been constructed from Xcc strain P20H in our laboratory. Plate assays for the extracellualr enzymes showed that pilD mutant exhibited the same levels of activity as those in the wild-type cells. To compare the N-terminal sequence, extracellular proteins from the culture supernatants of P20H and MC1220 (pilD::pSMP104) were separated in SDS-polyacrylamide gel electrophoresis, transblotted onto Nylon membrane, then three of the proteins (24, 28 and 35-kDa) were subjected to chemical determination of the N-terminal sequences. The results showed that each of the protein pairs has the same terminal ends. N-terminal sequencing also identified the 24, 28, and 35-kDa proteins as the conserved hypothetical protein (XCC0694 in the genome of Xcc strain ATCC33913), cellulose S (XCC3381), and pectate lyase II (XCC2815, designated as PelB herein), respectively. These results indicate that PilD is not the signal peptidase responsible for the N-terminus processing during secretion of these extracellular proteins.
In a separate experiment, SDS-PAGE showed that the amounts of extracellular PelB were significantly reduced in a mutant deficient in Clp, a transcription factor homologous to CRP (cyclic AMP-receptor protein), suggesting that expression of pelB is regulated by Clp. Because of the latter finding, pelB was further studied. The results indicate that 1) the pelB mutant of Xcc acquired by insertional mutation showed no change in plate assays for pectinolytic activity, indicating that PelB is not the major pectate lyase in Xcc, 2) in pathogenicity testing using pelB mutant, appearance of symptoms was delayed compared with that of the wild-type virulent strain, and 3) in transcriptional fusion (pelB-lacZ) assays, the promoter activities were found to be greatly reduced in clp and rpfF mutant; suggesting that transcription of the pelB gene is positively regulated by Clp as well as RpfF.
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