Functional anaiysis of the regulatory luxZ gene from Photobaterium leiognathi

• Main Author: 陳玉峰
• Other Authors: 林瑞文
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/56573762484448677296

碩士 === 國立中興大學 === 生物化學研究所 === 91 === Photobacterium leiognathi PL741 lux operon cloned in E. coli showed dim; it suggests that the specific regulatory gene(s) is required for P. leiognathi lux operon induction. P. leiognathi ATCC 25521 luxZ gene was cloned, by in trans complementation Lux-assays in vivo, it encoded the LuxZ protein which enables to enhance the PL741 lux operon in E. coli. LuxZ, a regulatory protein belonging to the FadR sub-family of GntR family, has a Mr ~25.8 kDa. Analysis of the [luxZ]R&R shows that the major promoter for the luxZ transcription is P*, and the [luxZ]R&R is possible to autoregulate by the secondary mRNA structures or trans-acting factors binding the UAS/OI,AS/OII sequences. Bioassays of the luxZ gene’s enhancement revealed that the LuxZ can enhance bioluminescence of PL741 lux operon and V. fischeri lux regulon [lux]R&R(PL), but functions weak for ATCC 25521 [luxZ]R&R(PR). In addition, the specific gene(s) which enhanced by the luxZ were selected from P. leiognathi ATCC 25521 genomic library. The LuxZ can regulate the [orf2]R&R and the intern promoter resided in the orf2 gene. Besides, the LuxZ can shift the orf2 transcription initiation sites by unknown mechanism. Glucose repression shows that the [orf2]R&R is also regulated by cAMP-CRP. Functional analysis of the cAMP-CRP and LuxZ enhancements elucidates that (1) the enhancements in PL741 [luxZ]R&R are independent, and additive. (2) The conservation of the cAMP-CRP and LuxZ conserved DNA binding sites determines the function of enhancement. The DNA-binding assays display that the LuxZ enhancement is related to the specific conserved DNA sequences (AT rich DNA repeats) and the convenient binding sites for RNA polymerase to transcript. The luxZ gene was induced at stationary phase, it suggested that the function of the LuxZ was concerned with stress-resistant.