Transcriptional analysis of pilBA2A1CD cluster in xanthomonas campestris pv. campestris

• Main Author: 黃培瑄
• Other Authors: 曾義雄
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/56470531185800389731

碩士 === 國立中興大學 === 分子生物學研究所 === 90 === AU56E, a clp mutant derived from Xanthomonas campestris pv. campestris strain 17, synthesizes no significant amounts of xanthan gum (an exopolysaccharide). In addition, this mutant is resistant to filamentous phage fLf due to the failure in phage adsorption. Furthermore, studies carried out in our laboratory have indicated that 1) fLf infection is through attachment to the type IV pilus, the primary receptor, 2) pilRSBA1A2CD gene cluster is essential for the normal biogenesis of pilus, and 3) biogenesis of the type IV pilus is regulated by Clp, a global transcription factor. In order to understand the expression and regulation of genes pilA1, pilA2, pilB, pilC, and pilD, the promoter activities of pilA1, pilB and pilC were measured in two types of transcriptional fusions: one with the promoter-less xylE-GmW cartridge and the other with the promoter-less lacZ as the reporter. In the former assays, the cartridge was inserted into the coding regions of the genes to be studied while in the latter assays a promoter region was cloned upstream of the lacZ gene. The results of xylE-GmW reporter assays showed that pilA1 promoter is drastically reduced in clp mutant, indicating that it is positively regulated by Clp. No effects were observed in the expression of pilB and pilC promoters. In lacZ fusion assays, the result indicated that 1) neither growth of each strains nor the activities of the promoters were affected by fLf infection, 2) expression of the pilA1 and pilC promoter was higher in the wild-type Xc17 than those in AU56E, indicating that that transcription of pilA and pilC gene requires Clp, and 3) the promoter activities of pllA2, pilB and pilD were not affected by the mutation of clp gene. Primer extension analysis showed that the transcription start site of pilB is the T located at 15 nt upstream from the ATG start codon, and that of pilC is the G located at 21 nt upstream from the ATG start codon. In addition, Northern blot analysis results suggest that pilA1, pilA2 and pilC are transcribed from different promoters.