Study Of Differential Proteomics Of Gram-positive Moderately Halophilic Bacteria Response To Osmotic Stress Conditions

Halobacillus dabanensis D-8~T, a Gram-positive moderately halophilic bacterium, was isolated from the saline deposits of Daban lake in the Xinjiang Uygur Autonomous Region of China. It can grow in the complex medium containing 0.5% to 23% salt with the optimum growth salinity ranging from 3% to 15%. To investigate the salt tolerant mechanisms of the Gram-positive moderately halophilic bacterium D-8~T, the differential proteome techniques was employed and the high resolution 2-D PAGE maps were obtained. About 700 protein spots with the molecular weight between 17.5~66kDa were visualized in the gels, which were spread in the gel of the isoelectric point 3.5 to 5.9. Plenty of high abundance proteins were mainly clustered in the narrow region with the isoelectric point between 4.5 and 5.0, but most of low abundance proteins were in the other region dispersedly. These indicate that the most proteins in the strain D-8~T would be acidic.Some proteins related to salt shock were identified by applying the proteome techniques to analyze the differential protein expression files of the strain D-8~T exposed to severe salt shock. Comparative analysis of the protein expression files was carried out between the strain incubated in the 1% salinity medium (18h) and the ones shocked immediately with high salinity (25%) for 5min and 50min, respectively. There were 74 protein spots expressed differently. Among them, the expression of 24 protein spots were increased markedly while 8 protein spots were induced by salt stress, and the other 42 protein spots were decreased obviously. After hyperosmotic challenge, 7 up-regulated protein spots were identified through MALDI-TOF/MS and MASCOT. They were involved in CheY-like receiver in signal transduction pathways, oxaloacetate decarboxylase and enolase in energy metabolism pathways, heat shock protein and molecular chaperone such as class I heat shock protein, ATPases with chaperone activity, ClpC ATPase et al, and anti-adversity protein such as enolase. It was the first time that ClpC ATPase was identified in Gram-positive moderately halophilic bacteria.To study differential expression protein files of moderately halophilic bacteria subjected to hypoosmotic shock, comparative analysis of the protein expression files was carried out between the strain incubated in the 20% salinity medium (18h) and the ones shocked immediately with low salinity (0%) for 5min and 50min, respectively. There were 39 protein spots expressed differently. Among them, the expression of 21 protein spots were up-regulated clearly involving 6 new induced protein spots and the other 18 protein spots were decreased obviously. After hypoosmotic stress, 5 up-regulated protein spots were identified through MALDI-TOF/MS and MASCOT. They were similar to chaperone protein DnaK, rod shape-determining protein, 5-enolpyruvoylshikimate- 3-phosphate synthase, penicillin-binding protein and ClpC ATPase, respectively. It was the first time that heat shock proteins were found to be induced in bacterium after a shift to low-osmolarity.By means of differential proteomic approach, differential expression protein files of moderately halophilic bacteria grew in different salinity medium was studied, and some proteins related to high salinity were identified. It was found that 133 protein spots expressed differently. In comparison with the sample in 1% salinity, the expression of 88 protein spots was up-regulated remarkably when the strain D-8~T responded to long-term high salt stress between 10% and 20%. Among them, 26 new protein spots were induced by high salt. In addition, 45 protein spots were depressed evidently, among which 20 protein spots disappeared in high salinity. From the results of mass spectrum analyzing and protein databases matching, when the strain D-8~T grew in high salt overexpressed proteins were shown as follows: related to energy-producing pathway, involved in the cell growth and propagation, response to many kinds of stress like global stress regulators and concerned with compatible solutes including trehalose and beta...