中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析
生物工程学报 Chin J Biotech 2008, March 25; 24(3): 395-400
journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061
cjb@im.ac.cn . 2008 Institute of Microbiology, CAS & CSM, All rights reserved
张薇1,2, 魏海雷3, 高洪文2, 黄国和1
1 华北电力大学能源与环境研究中心, 北京 102206
2 中国农业科学院北京畜牧兽医研究所, 北京 100094
3 中国科学院微生物研究所, 北京 100101
摘 要: 中度嗜盐菌Bacillus alcalophilus DTY1 分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1 菌株四氢嘧啶合成基因簇ectABC。ectA、ectB 和ectC 分别编码169、428 和132 个氨基酸的肽链, 分别与B.halodurans C-125 中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb 片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC 检测发现, 在1.0% NaCl 浓度下, 转化菌B. cereus Z-E 菌株生成70.1 mg/g 四氢嘧啶, 而在5.0%的NaCl 浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1 的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC 的表达受盐诱导。
关键词: 四氢嘧啶, Bacillus alcalophilus, 中度嗜盐菌
Cloning and Characterization of ectABC Cluster from Bacillus alcalophilus DTY1
Wei Zhang1,2, Hailei Wei3, Hongwen Gao2, and Guohe Huang1
1 Energy and Environmental Research Center, North China Electric Power University, Beijing 102206, China
2 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100094, China
3 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Abstract: Bacillus alcalophilus DTY1, one moderate halophytic bacterium isolated from saline soil in Loess Plateau of China, was characterized with efficient production of ectoine. In this study, the gene cluster ectABC taking in charge of biosynthesizing ectoine was cloned from the genomic library of strain DTY1. Nucleotide sequencing indicated that ectA, ectB and ectC were predicted to encode peptides of 169, 428 and 132 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC share 59%, 81% and 81% identity to 2,4-diaminobutyric acid acetyltransferase, 2,4-diaminobutyric acid transaminase and ectoine synthase of B. halodurans C-125, respectively. A fragment containing ectABC genes was introduced into B. cereus Z, which made the transgenic Z cells increased tolerance to salt, remarkably. HPLC analysis of ectoine in the transgenic Z cells revealed that 70.1 mg/g ectoine was detected in 1.0% NaCl medium and 118.6 mg/g ectoine in 5.0% NaCl medium. Furthermore, as the concentration of salt increased, transgenic Z cells accumulated more ectoine. These results suggest that ectoine is an important facet in B. alcalophilus DTY1 to high-osmolarity surroundings, and the expression of ectABC is induced by salt strength.
Keywords: ectoine, Bacillus alcalophilus, moderate halophytic bacteria
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