| Title | Identification and Characterization of Ectoine Biosynthesis Genes and Heterologous Expression of the ectABC Gene Cluster from Halomonas sp. QHL1, a Moderately Halophilic Bacterium Isolated from Qinghai Lake |
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| Author | Derui Zhu1,2, Jian Liu1, Rui Han3, Guoping Shen2, Qifu Long2, Xiaoxing Wei2, and Deli Liu1* |
| Address | 1Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan 430079, P. R. China, 2Research center of Basic Medical Sciences, Qinghai University Medical College, Xining 810016, P. R. China, 3Qinghai Academy of Agricultural Forestry Sciences, Qinghai University, Xining 810016, P. R. China |
| Bibliography | Journal of Microbiology, 52(2),139-147, 2014, |
| DOI | 10.1007/s12275-014-3389-5 |
| Key Words | Halomonas, compatible solutes, ectoine, ectABC gene cluster, cloning and expression |
| Abstract | The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ70-dependent promoter and a δ38-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain. |