Molecular Characterization of the Gene Encoding Threonine Dehydrogenase in Xanthomonas campestris

• Main Authors: Liu, Yi-Shiow, 劉怡秀
• Other Authors: Weng Shu-Fen
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/30728002205307088940

碩士 === 國立中興大學 === 分子生物研究所 === 85 === The tdh gene of Xanthomonas campestris pv. campestris 17 (Xc17) was cloned and sequenced. The gene product has a high degree of identity to the threonine dehydrogenase (TDH) of E. coli in amino acid sequence. Primer-extension analysis showed that the +1 site was located 42 bp upstream from the translational start codon of the tdh gene in Xc17. The putative -10 (TATAAT) and -35 (TGGCGC) regions were located bp -11 to -6 and bp -32 to -27 upstream of the transcriptional start site, respectively. Deletion analysis demonstrated that the -10 sequence, but not the -35 sequence was critical for the tdh promoter activity. Two putative Lrp (leucine-responsive regulatory protein)-binding sites were also located between bp -58 and -46 upstream and bp +2 and +14 downstream from the transcriptional start site, respectively. Presence of these sequences suggests that binding of Lrp may be involved in the regulation of tdh expression in Xc17. Similar to most of the X. campestris promoters, the tdh promoter expressed poorly in E. coli. A putative terminator, locating at 22 bp downstream of the translational stop codon, was functional in terminator probe assay in E. coli and X. campestris. These results suggest that transcription from the tdh promoter may produce a monocistronic mRNA. This prediction was confirmed by Northern-blot analysis which showed that the transcript indeed had a size similar to that of the tdh coding region. The activity of threonine dehydrogenase in the tdh mutant, constructed by gene replacement, was found to be regained in the presence of the cloned fragment containing the complete tdh gene sequence and its promoter.