Proteome Analysis of Halobacterium sp. NRC-1 Facilitated by the Biomodule Analysis Tool BMSorter

Abstract

To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms. Halobacterium species NRC-1 is an extremely halophilic archaeon containing a highly acidic proteome with a median pI of 4.9, a property that is essential to the maintenance of the solubility and function of the proteins in a high salinity environment of about 5 M salts (1, 2). Genome sequence analysis has revealed 2,630 putative protein-coding genes in the 2,571,010-bp genome (2). Among the predicted proteins, 1,658 can be matched to sequences in public databases. Of the matches, 1,067 are proteins of known or predicted function, and 591 are proteins of unknown function. The possession of a relatively small and completely sequenced genome, the availability of a full arsenal of genetic manipulation tools, and the relative ease of culture make Halobacterium sp. NRC-1 an attractive systems biology model organism of the domain Archaea (3-6).The genomes of Halobacterium species are extremely unstable (7-9). Early studies of Halobacterium sp. NRC-1 (also known as Halobacterium halobium) and the closely related Halobacterium salinarium discovered unusually high spontaneous mutation frequencies of 0.01% for the production of bacteriorhodopsin-or bacterioruberin-deficient phenotypes and a more striking 1% for partial or total gas vesicle-deficient phenotypes. The species is also noteworthy for the large number of insertion sequence elements that are harbored in this unstable genome. Molecular genetic analysis of the bacteriorhodopsin-and gas vesicle-deficient mutants established a relationship between transposable insertion sequence-mediated insertional inactivation or deletions of structural or regulatory genes and the high mutant rates (10 -16). Upon the completion of the genome sequence, DNA analysis revealed the presence of 91 copies of insertion sequence elements belonging to 12 families in the Halobacteri...

Halobacterium salinarum NRC-1 PeptideAtlas: Toward Strategies for Targeted Proteomics and Improved Proteome Coverage

“…Although all proteins from the arginine deiminase pathway, including ArcRABC, were previously detected25, here we have newly detected previously elusive components of the DMSO and phototrophic respiratory pathways DmsC and the regulator Bat (Table 3). Specifically, we were able to detect 183 unique peptides from 13 proteins involved in energy production via bacteriorhodopsin-mediated phototrophy by enriching for the purple membrane (Table 1: fractionation, ICAT, and bacteriorhodopsin IP gel band extraction experiments)31.…”

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples

“…This is especially true for the acidic proteome of Halobacterium, as we were able to identify 1181 total proteins by combining UVPD with other traditional MS/MS methods (Fig. 4)-the most proteins identified to date for this organism (to the best of our knowledge) (39,40). The 1181 identified proteins covered 45% of the 2630 predicted proteins from Halobacterium NRC-1 (41) and were identified without the use of labor-intensive sample prefractionation techniques.…”

T4-Like Genome Organization of the Escherichia coli O157:H7 Lytic Phage AR1

“…The viral structural proteins were treated with TCEP-HCl (Pierce, Rockford, IL) and digested with trypsin at 37°C overnight, similar to a previously described method (16). Trypsin-digested peptides were purified using a PepClean C 18 spin column (Pierce, Rockford, IL) and analyzed by LTQ Orbitrap hybrid mass spectrometers (Thermo Scientific, San Jose, CA).…”