Expression and Regulation of Xanthomonas campestris pv. campestris α-Amylase Gene

α-amylase, which hydrolyzes starch to a mixture of maltose, maltotriose and relatively small amount of glucose, is one of the extracellular proteins produced by Xanthomonas campestris pv. campestris. The gene encoding the α-amylase (amy) has been cloned from strain Xc11. The purpose of this study was to locate and analyze the promoter and the regulatory region of Xc α-amy from Xanthomonas campestris pv. campestris 11. To achieve this end, the upstream region of the amy gene was cloned and sequenced. The total length ofthe DNA sequenced was 1091 bps, which ranged from -836th to +255th bp relative to the translational initiation site (+1). The sequence analysis illustrated that the upstream region of α-amy contained a putative upstream activator-binding site ΩUAS, potential promoter -10/-35 sequences, two inverted repeats ΩI , ΩII, and Shine-Dalgarno sequence (SD). Sequence comparison result revealed that the -376th to -482th bp upstream of Xcα-amy showed extensive homolgy to the -229th to -123th bp of Xc putative sigma factor gene (rfaY). The 0.8 kb insert DNA fragment of PE800 carrying the putative promoter sequence was cloned into the promoter proving vector pFY7 which uses bacterial luciferase as the reporter, forming plasmid pAMY800. Xc17(pAMY800) was able toemit bioluminescence suggesting that the 0.8 kb insert DNA carried promoter sequences. To understand the effect of carbohydrates on the expression of α-amy promoter, Xc17(pAMY800) was incubated in minimal medium containing one of the eight different carbon sources. Results showed that expression of α-amy promoter could be induced by the presence of maltose, starch, glucose, and sucrose in the medium, but functioned poorly in the medium containing galactose, lactose, fructose, or glycerol medium.. The time course of luciferase activity expressed by Xc17(pAMY800) coincided with that of the amylase activity expressed by Xc17 grown in LB, maltose or galactose medium. However, it seemed that glucose would inactivate upon the enzyme activity of α- amylase, but not on the expression by α-amy promoter. To determine the border region of the Xc α-amy promoter and regulatory region, PCR technique and Bal31 exonuclease deletion system were used to construct the 5' deletion mutants; and methods of site-directed mutagenesis and hydroxylamine random mutagenesis were used to generate mutation clones in this regulatory region. Totally 14 mutation clones were selected and assayed by the system using expression of bioluminescence as as a function of promoter activity. Results showed that sequences around the ΩUAS palyed an important role in maltose induction and galactose repression. The minimal 5' end for α-amy regulation was located around -187. And the 5' upstream sequence essential for expression of Xc α-amy promoterefficiently was located around -133th to -119th bp, sequence around the putative -35 region. In addition, our data also showed that the amy promoter can not function efficiently in E. coli