Identification and Functional Analysis of the luxZ Gene that Enhances Bioluminescence of the lux Operon from Photobacterium leiognathi

• Main Authors: Lu, Han-Chang, 盧漢章
• Other Authors: Juey-Wen Lin
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/14398463009325596588

碩士 === 國立中興大學 === 農業生物科技學研究所 === 84 === Photobacterium leiognathi 的螢光調控機制仍不甚瞭解，其螢光表現 顯受調控。本研究嘗試選殖可調控 P. leiognathi lux operon 之基因； 以 in trans com-plementation 之方法建立 P. leiognathi gene library， 選殖 lux operon 的 regula-tory gene 或可 enhance lux operon 螢光表現之基因。經選殖得一質體 pHC4 明顯 enhances P. leiognathi lux operon 之螢光表現；pHC4 具一 2.1 kb P. leiognathi genomic DNA，經 DNA 序列定序與分析，此段 DNA 序列的基因排列如下 ： pkI-ter--- R&R-luxZ其中一完整基因，定名為 luxZ gene 為可調 控 lux operon 之基因。luxZ gene encodes 一 221 amino acid residues 之蛋白質，其分子量 Mr 25 kDa，經氨基酸分析比對其與 Pseudomonas strain LB400 之 orf0 基因類似，可能為一屬於 GntR family 之 regulatory protein。序列分析發現在 luxZ gene 上游具有 調控區域 (Regulatory Region, R&R)，以 promoter-proving vector/ terminator-proving vector 及 bioluminoassay in vivo 之方法，可確 認此區域為 luxZ 及其相關基因的 regu-latory region R&R, 具有 upstream activating seguence WUAS, dual promoters, operator。 經 maxicell 方法分析，可知選殖的 luxZ gene 可主導 LuxZ protein 的合 成。調節蛋白 LuxZ 經功能分析後，得知其並非 P. leiognathi lux operon 之專一性的調節基因 (regulatory gene)。luxZ gene enhances P. leiognathi lux operon 之螢光表現，其功能可能與 cAMP-CRP binding site 及 cAMP-CRP complex binding 有關；luxZ gene 應是 positive regulatory gene。Glucose repression 明顯的抑制 LuxZ enhances P. leiognathi lux operon 的螢光表現的作用。 The specific gene designated as luxZ gene that enhances bioluminescence of the lux operon from Photobacterium leiognathi PL741 in E. coli was cloned and identified. Nucleotide sequence of the regulatory region R&R with the luxZ gene has been determined, and the amino acid sequence of the protein encoded by the luxZ gene is deduced. Functional analysis elucidates that the regulatory region R&R, which inludes upstream activator- binding site WUAS, dual -10/-35 canonical promoters, putative operator and Shine-Dalgarno (SD) sequence, indeed functions for gene expression and regulation of the luxZ and related genes. The LuxZ protein has a calculated Mr of 25,807 and comprises 221 amino acid residues. The luxZ gene, which apparently enhances bioluminescence of the lux operon from P. leiognathi in E. coli, was identified as regulatory gene for the lux operon by in trans complementation bioluminoassays in vivo. The LuxZ protein was identified as the putative transcriptional regulator by significant ssimilarity with the hypothetical transcriptional regulator encoded by orf0 genefrom Pseudomonas sp. LB400. The LuxZ protein, which functions as positive regulator for the lux operon from P. leiognathi to enhance bioluminescence, is likely to be the DNA-binding protein related to the GntR family of transcripti-onal regulators. In addition, partial 3'-end nucleotide sequence of the pkI gene from P. leiognathi PL741 has been cloned and determined, and the amino acid sequence of pyruvate kinase I encoded by the pkI gene is deduced. Pyruvate ki-nase I is the key enzyme, which converts phosphoenol pyruvate to pyruvate, for glycolysis. Alignment and comparison of pyruvate kinases I from P. leiogathi and E. coli shows that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiogathi. The gene order of the pkI and luxZ genes is pkI ter->-R&R-luxZ-> (R&R: regula-tory region; ter, terminator), whereas the R&R is the regulatory region for the luxZ gene, and ter is the transcriptional terminator for the pkI and related genes. It clearly elicits that pkI gene and luxZ gene belong to two diffenent operons. Functional ana-lysis confirms that the potential hair-pin loop WT is the transcriptional terminator for the pkI and related genes. It suggests that the pkI and related genes are simply linked to the luxZ gene in genome.