SummaryA global view of the biology of the cold-adapted archaeon Methanococcoides burtonii was achieved using proteomics. Proteins specific to growth at 4 ∞ ∞ ∞ ∞ C versus T opt (23 ∞ ∞ ∞ ∞ C) were identified by mass spectrometry using the draft genome sequence of M. burtonii . mRNA levels were determined for all genes identified by proteomics, and specific enzyme assays confirmed the protein expression results. Key aspects of cold adaptation related to transcription, protein folding and metabolism, including specific roles for RNA polymerase subunit E, a response regulator and peptidyl prolyl cis/trans isomerase. Heat shock protein DnaK was expressed during growth at T opt , indicating that growth at 'optimal' temperatures was stressful for this cold-adapted organism. Expression of trimethylamine methyltransferase involves contiguous translation of two open reading frames, which is likely to result from incorporation of pyrrolysine at an amber stop codon. Thermal regulation in M. burtonii is achieved through complex gene expression events involving gene clusters and operons, through to protein modifications.

Direct analysis of membrane lipids by liquid chromatography-electrospray mass spectrometry was used to demonstrate the role of unsaturation in ether lipids in the adaptation of Methanococcoides burtonii to low temperature. A proteomics approach using two-dimensional liquid chromatography-mass spectrometry was used to identify enzymes involved in lipid biosynthesis, and a pathway for lipid biosynthesis was reconstructed from the M. burtonii draft genome sequence. The major phospholipids were archaeol phosphatidylglycerol, archaeol phosphatidylinositol, hydroxyarchaeol phosphatidylglycerol, and hydroxyarchaeol phosphatidylinositol. All phospholipid classes contained a series of unsaturated analogues, with the degree of unsaturation dependent on phospholipid class. The proportion of unsaturated lipids from cells grown at 4°C was significantly higher than for cells grown at 23°C. 3-Hydroxy-3-methylglutaryl coenzyme A synthase, farnesyl diphosphate synthase, and geranylgeranyl diphosphate synthase were identified in the expressed proteome, and most genes involved in the mevalonate pathway and processes leading to the formation of phosphatidylinositol and phosphatidylglycerol were identified in the genome sequence. In addition, M. burtonii encodes CDP-inositol and CDP-glycerol transferases and a number of homologs of the plant geranylgeranyl reductase. It therefore appears that the unsaturation of lipids may be due to incomplete reduction of an archaeol precursor rather than to a desaturase mechanism. This study shows that cold adaptation in M. burtonii involves specific changes in membrane lipid unsaturation. It also demonstrates that global methods of analysis for lipids and proteomics linked to a draft genome sequence can be effectively combined to infer specific mechanisms of key biological processes.

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Genome sequence data of the cold-adapted archaeon, Methanococcoides burtonii, was linked to liquid chromatography-mass spectrometry analysis of the expressed-proteome to define the key biological processes functioning at 4 degrees C. 528 proteins ranging in pI from 3.5 to 13.2, and 3.5-230 kDa, were identified. 133 identities were for hypothetical proteins, and the analysis of these is described separately (Goodchild et al. manuscript in preparation). DNA replication and cell division involves eucaryotic-like histone and MC1-family DNA binding proteins, and 2 bacterial-like FtsZ proteins. Eucaryotic-like, core RNA polymerase machinery, a bacterial-like antiterminator, and numerous bacterial-like regulators enable transcription. Motility involves flagella synthesis regulated by a bacterial-like chemotaxis system. Lsmalpha and Lsmgamma were coexpressed raising the possibility of homo- and hetero-oligomeric complexes functioning in RNA processing. Expression of FKBP-type and cyclophilin-type peptidyl-prolyl cis-trans isomerases highlights the importance of protein folding, and novel characteristics of folding in the cold. Thirteen proteins from a superoperon system encoding proteasome and exosome subunits were expressed, supporting the functional interaction of transcription and translation pathways in archaea. Proteins involved in every step of methylotropic methanogenesis were identified. CO(2) appears to be fixed by a modified Calvin cycle, and by carbon monoxide dehydrogenase. Biosynthesis involves acetyl-CoA conversion to pyruvate by a non-oxidative pentose phosphate pathway, and gluconeogenesis for the conversion of pyruvate to carbohydrates. An incomplete TCA cycle may supply biosynthetic intermediates for amino acid biosynthesis. A novel finding was the expression of Tn11- and Tn12-family transposases, which has implications for genetic diversity and fitness of natural populations. Characteristics of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

The oceans of the world are nutrient-limited environments that support a dynamic diversity of microbial life. Heterotrophic prokaryotes proliferate in oligotrophic regions and affect nutrient transformation and remineralization thereby impacting directly on the all marine biota. An important challenge in studying the microbial ecology of oligotrophic environments has been the isolation of ecologically important species. This goal has been recognized not only for its relevance in defining the dynamics of community composition, but for enabling physiological studies of competitive species and inferring their impact on the microbial food web. This review describes the successful isolation attempts of the ultramicrobacterium, Sphingopyxis alaskensis (formerly described as Sphingomonas alaskensis) using extinction dilution culturing methods. It then provides a comprehensive perspective of the unique physiological and genetic properties that have been identified that distinguish it from typical copiotrophic species. These properties are described through studies of the growth phase and growth rate control of macromolecular synthesis, stress resistance and global gene expression (proteomics). We also discuss the importance of integrating ecological and physiological approaches for studying microorganisms in marine environments.