[Cloning of the ectoine biosynthesis gene ectABC from Halomonas sp. BYS-1 and salt stressed expression in Escherichia coli].
Ectoine was the main compatible solute of moderately halophilic bacteria. In order to clone the ectABC gene which involved in the ectoine biosynthesis pathway from total DNA of moderately halophilic bacteria Halomonas sp. BYS-1, firstly a 750bp fragment of ectABC gene was amplified by PCR using combinations of forward primers and reverse primers designed according to the ectABC genes of Halomonas elongata 2851T and Halomonas elongata DSM3043. Then the upstream and downstream sequences of the 750bp fragment were amplified by SEFA PCR (SElf-Formed Adaptor PCR), a new PCR method amplified relatively long flanking sequences from tagged sequences in a simple way without enzyme excision and ligation. The 3532bp fragment include 2423bp ectABC, 980bp upstream sequences and 129bp downstream sequences were cloned from Halomonas sp. BYS-1 using a pair of conserved primers designed according to acquired sequences by SEFA PCR. The GenBank accession number of the 3532bp fragment is DQ017757. ORF analysis revealed that ectA, ectB, ectC cluster to an operon, the size of ectA, ectB, and ectC were 573bp, 1251bp and 387bp respectively. The predicted molecular masses of the encoded proteins were 21.1kDa (191 amino acids, EctA), 45.7 kDa (417 amino acids, EctB), and 14.5 kDa (129 amino acids, EctC) respectively. The 3532bp fragment was ligated to the MCS site of vector pUC19 and transformed E. coli DH5alpha to construct E. coli (pUC19ECT). Transformant E. coli (pUC19ECT) could synthesis ectoine under salt stress, the intracellular ectoine level were 7.1, 19.4 and 32.3 micromol/(g x dry x wt) when the salinities of the mediums were 0, 0.4 and 0.8mol/L sodium chloride respectively. But the accumulation of ectoine could not promote the growth of E. coli (pUC19ECT)under high salinity.
Key Lab of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Microbiology Department, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China. hejian@njau.edu.cn
Jian
J
He
Xing
X
Huang
Li-feng
LF
Gu
Jian-dong
JD
Jiang
Shun-peng
SP
Li
D000599:Amino Acids, Diamino / Q000138:chemical synthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D020632:Halomonas / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012965:Sodium Chloride / Q000378:metabolism
D000599:Amino Acids, Diamino; D001426:Bacterial Proteins; D017931:DNA Primers; D012965:Sodium Chloride; C045628:ectoine