Summary auto-generated
This study identified and characterized genes for ectoine biosynthesis from the halophilic bacterium Marinococcus halophilus. Researchers cloned a 4.4 kb DNA fragment containing four open reading frames into Escherichia coli, enabling osmoregulated ectoine synthesis in response to increased medium salinity. Sequencing and functional analysis revealed three genes essential for ectoine production: ectA (encoding L-2,4-diaminobutyric acid acetyltransferase), ectB (encoding L-2,4-diaminobutyric acid transaminase), and ectC (encoding L-ectoine synthase). Ectoine accumulated to 1 mmol per gram dry weight at 5% NaCl, demonstrating that the genes were not only expressed but also osmoregulated in E. coli. DNA sequences upstream of ectA were shown to contain osmotic regulatory elements. The work provides the first molecular characterization of compatible solute biosynthesis genes from a true halophile and demonstrates that osmoregulatory mechanisms are conserved across phylogenetically distant bacteria.
Key findings
- Three genes (ectA, ectB, ectC) encode enzymes catalyzing the three-step biosynthetic pathway for ectoine from aspartate-β-semialdehyde
- Ectoine synthesis in E. coli was osmoregulated, with cytoplasmic concentration increasing from 0 to 1 mmol/g dry weight as medium salinity increased from 0% to 5% NaCl
- Regulatory sequences controlling osmotic expression are located upstream of ectA, approximately 150 bp from the first open reading frame
- The ectoine biosynthesis genes from the halophile M. halophilus functioned with osmotic regulation when expressed in the non-halophile E. coli, suggesting conservation of osmoregulatory mechanisms across distant bacteria
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Abstract
The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2- methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4.4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.