Molecular characterization of orf482 required for the synthesis of Xanthan polysaccharide in Xanthomonas campestris pv. campestris

• Main Authors: Chang, chai-ming, 張家銘
• Other Authors: Yi-Hsiung Tseng, Fu-shyan Wen
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/98032256597646166061

碩士 === 國立中興大學 === 植物學系 === 86 === Abstract: The gram-negative bacterium Xanthomonas campestris pv. campestris is an important plant pathogen, causing black rot in crucifers worldwide resulting in tremendous loss. It is also a producer of xanthan gum, a substance which has a variety of applications in petroleum production, cosmetics and food industries. We were interested in expression and regulation of the genes involved in xanthan gum synthesis. We have previoumutagenesis. Using pulsed-field gel electrophoresis (PFGE) and Southern hybridization, the mutations leading to the non-mucoid phenotypes have been located at eight loci in the Xc17 chromosome, which were named eps1, eps2, eps3, eps4, eps5, eps6, eps7 and eps8, respectively. This study focused on the non-mucoid mutant AY61E. One recombinant cosmid, pEXO40, was cloned from the genomic bank of Xc17. After deletion mapping, a smallest clone, pR2-2, was obtained which contained a 2.2-kb insert. Mucoid phenotype was restored to AY61E harboring pR2-2, indicating that the pR2-2 insert contained the gene responsible for the mutation in AY61E. Nucleotide sequence analysis of the pR2-2 insert revealed an open reading frame (orf482) able to encode a polypeptide with a calculated molecaa weight of 54,013 dal. The deduced amino acid sequence of the C-terminal 153 residues of ORF482 exhibited 99% identity to the gene 1 of X. campestris pv. campestris strain. In Northern hybridization of the total RNA extracted from Xc17, only one transcript with the size similar to that of the orf482 coding region (1.5 kb) was detected. Primer extension indicated the transcription start site to be the base T at 232 nt upstream from the orf482 codon region. A 458-bp PstI fragment before the orf482 start cod was found to efficiently express the lacZ reporter when cloned as a transcriptional fusion unit in the promoter-proving vector pFY13-9. Expression of the pR2-2 insert in vitro using E. coli S30 transcription/translation system produced a protein of 54 kDa which is similar to the MW deduced from nucleotide sequence. The promoter of gum operon, a cluster of 12 genes required for xanthan synthesis, cloned as a transcriptional fusion expressed normally in AY61E indicating that its expression is not regulated bORF482. AY61E exhibited higher resistance to chloramphenicol and salt. The amounts of the xanthan gum produced by AY61E and AY61E(pR2-2) were about 29.6% and 54.6%, respectively, of that produced by Xc17. AY61E showed attenuated virulence in causing black rot in chinese cabbage and pakchoi. The transcription direction of orf482 was determined to be counterclockwise on the Xc17 chromosome map.