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

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 δ(70)-dependent promoter and a δ(38)-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.

Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system

“…The structure of the ectoine gene cluster in strain ZD1 was identical to those of some Firmicutes and Proteobacteria species ( Halomonas sp. QHL1 [36], Marinococcus halophilus [37], and Bacillus alcalophilus DTY1 [11]. Expression of proteins (EctA, EctB, and EctC) in strain ZD1 grown under saline conditions was confirmed using SDS-PAGE analysis.…”

New Alpiniamides From Streptomyces sp. IB2014/011-12 Assembled by an Unusual Hybrid Non-ribosomal Peptide Synthetase Trans-AT Polyketide Synthase Enzyme

“…Desferrioxamine B, a high-affinity iron chelator (siderophore), is important for scavenging ferric iron as a nutrient for the cells ( Barona-Gómez et al, 2004 ). Ectoine, another common metabolite, is an osmoprotectant ( Zhu et al, 2014 ). The function of melanin, a black pigment, that is produced by many types of bacteria, is still under discussion ( Woo et al, 2010 ).…”

Draft Genome Sequence of Halomonas elongata Strain K4, an Endophytic Growth-Promoting Bacterium Enhancing Salinity Tolerance In Planta

“…The annotation of H. elongata strain K4 resulted in 2,354 ORFs, 5 rRNAs, 59 tRNAs, and 39 ncRNAs. Analysis of the genome indicated the presence of a single copy of the ectABC operon (14) involved in the biosynthesis of ectoine. The genome coded for the three main enzymes involved in the ectoine pathway, namely, L-2,4-diaminobutyric acid acetyltransferase (EctA) (EC: 2.3.1.178), L-2,4-diaminobutyric acid aminotransferase (EctB) (EC: 2.6.1.76), and L -ectoine synthase (EctC) (EC: 4.2.1.108).…”