Abstract:
The present study investigated the osmoadaptation strategies adopted by Brevibacterium sp. K11IcPPYGO002, a halotolerant novel strain isolated from the coastal dunes of Keri, Goa. Whole-genome sequencing revealed a genome size of 4,140,682 bp with a GC content of 63.97 percent. Genome annotation identified the ectoine/hydroxyectoine biosynthetic pathway, represented by the genes ask-asd, ectABC, and ectD, as well as the uptake system ehuABCD. The genes responsible for the biosynthesis of glutamate (gdhA), proline (proABC), and glycine betaine (betI and betABC) were detected. Trehalose and mannitol biosynthesis were indicated by the presence of genes otsAB and mtlK, respectively. Spermidine and putrescine synthesis were evidenced by speABC genes, along with the transport system (potABCD, potE, spuE). The genome harboured solute transporters (betT, betP, ectP, proP, opuA, opuC, gltT, proVWX), ion transporters, and osmoregulatory two-component systems (mtrA/B, kdpD/E), known to assist in salt tolerance. Functional validation of genomic data through LCMS confirmed the presence of intracellular compatible solutes (ICS) such as glutamic acid (146.20 [M-H]-, 147.90 [M+H]+), ectoine (142.90 [M+H]+), hydroxyectoine (158.90 [M+H]+), proline (116 [M+H]+), hydroxyproline (131.90 [M+H]+), choline (104 [M+H]+), glycine betaine (118 [M+H]+), dimethylsulfoniopropionate (135.90 [M]+), spermidine (145.90 [M+H]+), putrescine (111.90 [M+Na]), mannitol (182.90 [M+H]+) and trehalose (180.80 [C₆H₁₃O₆⁺]). LCMS-MRM demonstrated osmolarity-dependent increase in intracellular ectoine and hydroxyectoine, with ectoine peaking at 12 percent NaCl (25011.60 plus-minus 1852.69 ng/mg CDW) and hydroxyectoine at 16 percent NaCl (45.12 plus-minus 1.64 ng/mg CDW). STRING network analysis indicated coordinated ectoine biosynthesis. These findings provide genomic and metabolic insights into salt-stress adaptation in Brevibacterium sp. K11ICPPYGO002.
