This report summarizes the progress made on the title project during the grant period. We developed a new classification of two-dimensional separations based on the observation that separations can be made in time or in space. Thus, two-dimensional separations can be made in time×time, space×space, space×time, or time×space. The two successive separations must use two different modes of chromatography that afford uncorrelated or weakly correlated patterns of retention factors for the components of the samples analyzed. Our attention was mainly focused on the separation of protein digests, particularly, on those of the digests of myoglobin and bovine serum albumin as model systems and extremely efficient temporal separations were developed. We also designed and constructed new instruments to carry out space×space separations (True Bidimensional Chromatography, HPLC2 or spacial separations) and time×space separations (a new hybrid combination of a temporal and a spacial separation that we designed).

Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- …

The project investigated fundamental aspects of carbon metabolism and genetics in the methane-producing archaeon Methanococcus maripaludis. The project yielded 23 peer-reviewed publications and five reviews from 1997-2007. PDFs of the peer-reviewed publications are included in the next section. Some papers of special interest are listed below. The pathway of pyruvate biosynthesis was elucidated by a combination of biochemical and physiological studies. This work characterized the very oxygen sensitive pyruvate oxidoreductase and showed that the enzyme was irreversible under physiological conditions. Evidence for the flow of electrons from the energy coupling hydrogenase b (Ehb) was presented. These results were published in the following papers. Yang, Y.L., J.N. Gluska, and W.B. Whitman (2002) Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis. Arch. Microbiol. 178: 493-498. Lin, W.C., Y.L. Yang, and W.B. Whitman (2003) The anabolic pyruvate oxidoreductase from Methanococcus maripaludis. Arch. Microbiol. 179: 444-456. Lin, W., and W.B. Whitman (2004) The importance of porE and porF in the anabolic pyruvate oxidoreductase of Methanococcus maripaludis. Arch. Microbiol. 181: 68-73. Porat, I., W. Kim, E.L. Hendrickson, Q. Xia, Y. Zhang, T. Wang, F. Taub, B.C. Moore, I.J. Anderson, M. Hackett, J.A. Leigh, and W.B. Whitman (2006) Disruption of the Ehb hydrogenase operon limits …