Molybdate is transported in bacteria by a high-affinity transport system composed of a periplasmic binding protein, an integral membrane protein, and an energizer protein. These three proteins are coded by modA, modB, and modC genes, respectively. The ModA, ModB, and ModC proteins from various organisms (Escherichia coli, Haemophilus influenzae, Azotobacter vinelandii, and Rhodobacter capsulatus) are very similar. The lowest Km value reported for molybdate in the molybdate transport process is approximately 50 nM. In a mod mutant, molybdate is transported by the sulfate transport system or by a nonspecific anion transporter. Molybdate transport is tightly coupled to utilization in E. coli and Klebsiella pneumoniae, while other dinitrogen-fixing organisms appear to have a molybdenum storage protein. In all organisms studied so far, molybdate transport genes are regulated by a repressor protein, ModE. The ModE-molybdate complex binds to the sequences TAYAT (Y = T or C) in the operator/ promoter region in E. coli and prevents transcription of the modABCD operon. The ModE-molybdate complex binds to DNA as a homodimer in E. coli and possibly in other organisms as well. In R. capsulatus, however, two ModE homologues (MopAB proteins) are required for repression.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S 0 ). Growth rates were highest on media containing peptides and S 0 , with or without maltose. Growth did not occur on the peptide medium without S 0 . S 0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S 0 with or without maltose were the same, suggesting that S 0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S 0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S 0 -reducing enzyme in this organism and the mechanism of the S 0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.Hyperthermophiles are microorganisms that grow optimally at 80°C and above (46,47). Virtually all of them are strict anaerobes, and most are heterotrophs. All of the heterotrophs utilize peptides as a carbon source, and most use elemental sulfur (S 0 ) as a terminal electron acceptor leading to H 2 S production. The most studied of the S 0 -reducing, heterotrophic hyperthermophiles are species of Pyrococcus. Most of these organisms only utilize peptide-related substrates as a carbon source and show no significant growth in the absence of S 0 (9,12,19,36). Notable exceptions are Pyrococcus furiosus, P. woesei, and P. glycovorans, which are capable of metabolizing poly-and oligosaccharides, as well as peptides (2, 4, 10). P. furiosus and P. woesei can also grow to high cell densities in the absence of S 0 . The pathways of peptide and carbohydrate metabolism have been well studied in P. furiosus (1, 7). Glycolysis appears to occur via a modified Embden-Meyerhof pathway (Fig. 1) (22,35). This pathway is unusual in that the hexose kinase and phosphofructokinase steps are dependent on ADP rather than ATP, and a novel tungsten-containing enzyme termed glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) replaces the expected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase. Amino acid catabolism in P. furiosus is thought to involve four distinct 2-keto acid oxidoreductases that convert transaminated amino acids into their corresponding coenzyme A (CoA) derivatives (Fig. 2) (3,15,31,32). These CoA derivatives, together with acetylCoA produced from glycolysis via pyruvate...

In both bacteria and archaea, molybdate is transported by an ABC-type transporter comprising three proteins, ModA (periplasmic binding protein), ModB (membrane protein) and ModC, the ATPase. The modABC operon expression is controlled by ModE-Mo. In the absence of the high-affinity molybdate transporter, molybdate is also transported by another ABC transporter which transports sulfate/thiosulfate as well as by a nonspecific anion transporter. Comparative analysis of the molybdate transport proteins in various bacteria and archaea is the focus of this review.

One of the major unanswered questions in quantitative proteomics is that of dynamic protein turnover in the cell. Here we present a new approach to quantitative proteomics that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectrometry, stable isotope labeling is employed to calculate a relative synthesis/degradation ratio that reflects the relative rate at which 13C is incorporated into individual proteins in the cell. This synthesis/degradation ratio calculation is based on a Poisson distribution model that is designed to support high-throughput analysis. Protein separation and analysis is accomplished by utilizing one-dimensional SDS-PAGE gel electrophoresis followed by cutting the gel into a series of bands for in-gel digestion. The resulting peptide mixtures are analyzed via solid-phase MALDI LC-MS and LC-MS/MS using a tandem time-of-flight mass spectrometer. A portion of the soluble protein fraction from an E. coli K-12 strain was analyzed with synthesis/degradation ratios varying from approximately 0.1 to 4.4 for a variety of different proteins. Unlike other quantitative techniques, synthesis/degradation ratio mass spectrometry requires only a single cell culture to obtain useful biological information about the processes occurring inside a cell. This technique is highly amenable to shotgun proteomics-based approaches and thus should allow relative turnover measurements for whole proteomes in the future.