ABSTRACT: BACKGROUND:Halobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach. RESULTS:A modified 2-D gel electrophoretic procedure, employing IPG strips in the range of pH 3-6, enabled improved separation of acidic proteins relative to previous techniques. Combining experimental data from 2-D gel electrophoresis with available genomic information, allowed the identification of at least 30 cellular proteins involved in many cellular functions: stress response and protein folding (CctB, PpiA, DpsA, and MsrA), DNA replication and repair (DNA polymerase A alpha subunit, Orc4/CDC6, and UvrC), transcriptional regulation (Trh5 and ElfA), translation (ribosomal proteins Rps27ae and Rphs6 of the 30 S ribosomal subunit; Rpl31eand Rpl18e of the 50 S ribosomal subunit), transport (YufN), chemotaxis (CheC2), and housekeeping (ThiC, ThiD, FumC, ImD2, GapB, TpiA, and PurE). In addition, four gene products with undetermined function were also identified: Vng1807H, Vng0683C, Vng1300H, and Vng6254. To study the heat shock response of Halobacterium NRC-1, growth conditions for heat shock were determined and the proteomic profiles under normal (42 degrees C), and heat shock (49 degrees C) conditions, were compared. Using a differential proteomic approach in combination with available genomic information, bioinformatic analysis revealed five putative heat shock proteins that were upregulated in cells subjected to heat stress at 49 degrees C, namely DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2. CONCLUSION:The modified 2-D gel electrophoresis markedly enhanced the resolution of the extremely acidic proteome of Halobacterium NRC-1. Constitutive expression of stress proteins and chaperones help the organism to adapt and survive under extreme salinity and other stress conditions. The upregulated expression pattern of putative chaperones DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2 under elevated temperature clearly suggests that Halobacterium NRC-1 has a sophisticated defense mechanism to survive in extreme environments.
