Cloning and analysis of lon gene in Xanthomonas campestris pv campestris

• Main Authors: Tai Wei Hsia, 夏代威
• Other Authors: Yi-Hsiung Tseng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/25435732332034877119

碩士 === 國立中興大學 === 分子生物學研究所 === 89 === Xanthomonas campestris pv. campestris is a gram-negative, rod-shaped and monotrichously flagellated plant pathogenic bacterium. The chromosome of X. campestris pv. campestris has a G + C content more then 60%. It produces copious amounts of xanthan gum, which has been implicated to associate with the virulence of the bacterium. Biosynthesis of xanthan gum involves multiple steps and a multitude of enzymes. Recently, our laboratory has localized the genes involved in xanthan synthesis at eight loci on the X. campestris pv. campestris 17 (Xc17) chromosome map. In E. coli, lon gene mutations made cell mucoid, UV sensitive and long form cell. In this study, I employed a homologous recombination strategy to clone the of lon gene from the Xc17 chromosome . pOKRV carrying the DNA fragment of complete lon gene as thus obtained. Sequence analysis revealed 3955 bp containing two ORFs. The lon gene, nt 721 to nt 3189; encodes a protein that has 823 amino acids with a molecule weight of 90,631.27. The Lon protease deduced from the Xc17 possesses 65% similarity to that of E. coli and X. fastidiosa. Two motifs include the gene are ATP/GTP binding motif and ATP-dependent serine protease active site. The hupb, nt 3307 to 3680; encodes a protein 90 amino acids with a molecule weight of 9327.13, whose product is named Histone-like DNA binding protein. The sequence similarity of hupb is more then 65% with E. coli and P. fluorescens. A motif, the bacterial histone-like DNA-binding proteins signature motif, is present in the deduced HupB protein. Mutants of lon were constructed by insertional mutagenesis. No differences were found in colony morphology, pathogenicity, growth rate and UV-sensitivity between the mutant and the wild type cells