Tandem mass tag-based quantitative proteomics reveals osmotic adaptation mechanisms in Alkalicoccus halolimnae BZ-SZ-XJ29 T , a halophilic bacterium with a broad salinity range for optimal growth.

The moderate halophilic bacterium Alkalicoccus halolimnae BZ-SZ-XJ29 exhibits optimum growth over a wide range of NaCl concentrations (8.3-12.3%, w/v; 1.42-2.1 mol L ). However, its adaptive mechanisms to cope with high salt-induced osmotic stress remain unclear. Using TMT-based quantitative proteomics, the cellular proteome was assessed under low (4% NaCl, 0.68 mol L NaCl, control (CK) group), moderate (8% NaCl, 1.37 mol L NaCl), high (12% NaCl, 2.05 mol L NaCl), and extremely high (16% NaCl, 2.74 mol L NaCl) salinity conditions. Digital droplet PCR confirmed the transcription of candidate genes related to salinity. A. halolimnae utilized distinct adaptation strategies to cope with different salinity conditions. Mechanisms such as accumulating different amounts and types of compatible solutes (i.e., ectoine, glycine betaine, glutamate, and glutamine) and the uptake of glycine betaine and glutamate were employed to cope with osmotic stress. Ectoine synthesis and accumulation were critical to the salt adaptation of A. halolimnae. The expression of EctA, EctB, and EctC, as well as the intracellular accumulation of ectoine, significantly and consistently increased with increasing salinity. Glycine betaine and glutamate concentrations remained constant under the four NaCl concentrations. The total content of glutamine and glutamate maintained a dynamic balance and, when exposed to different salinities, may play a role in low salinity-induced osmoadaptation. Moreover, cellular metabolism was severely affected at high salt concentrations, but the synthesis of amino acids, carbohydrate metabolism, and membrane transport related to haloadptation was preserved to maintain cytoplasmic concentration at high salinity. These findings provide insights into the osmoadaptation mechanisms of moderate halophiles and can serve as a theoretical underpinning for industrial production and application of compatible solutes.