Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase. A member of a new class of Mg(2+)-dependent acid phosphatases.

Coenzyme M (CoM; 2-mercaptoethanesulfonic acid) is the terminal methyl carrier in methanogenesis. Methanogenic archaea begin the production of this essential cofactor by sulfonating phosphoenolpyruvate to form 2-phospho-3-sulfolactate. After dephosphorylation, this precursor is oxidized, decarboxylated and then reductively thiolated to form CoM. A thermostable phosphosulfolactate phosphohydrolase (EC 3.1.3.-) catalyzing the second step in CoM biosynthesis, was identified in the hyperthermophilic euryarchaeon Methanococcus jannaschii. The predicted ORF MJ1140 in the genome of M. jannaschii encodes ComB, a Mg2+-dependent acid phosphatase that is specific for 2-hydroxycarboxylic acid phosphate esters. Recombinantly expressed purified ComB efficiently hydrolyzes rac-2-phosphosulfolactate, (S)-2-phospholactate, phosphoglycolate and both enantiomers of 2-phosphomalate. In contrast to previously studied phosphoglycolate phosphatases, ComB has a low pH optimum for activity, a narrow substrate specificity and an amino acid sequence dissimilar to any biochemically characterized protein. Like other phosphatases that function via covalent phosphoenzyme intermediates, ComB can catalyze a transphosphorylation reaction. Homologs of comB are identified in all available cyanobacterial genome sequences and in genomes from phylogenetically diverse bacteria and archaea; most of these organisms lack homologs of other CoM biosynthetic genes. The broad and disparate distribution of comB homologs suggests that the gene has been recruited frequently into new metabolic pathways.

S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes.

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed in E. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the M. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.

An archaeal genomic signature.

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

Identification of a highly diverged class of S-adenosylmethionine synthetases in the archaea.

S-adenosylmethionine is the primary alkylating agent in all known organisms. ATP:L-methionine S-adenosyltransferase (MAT) catalyzes the only known biosynthetic route to this central metabolite. Although the amino acid sequence of MAT is strongly conserved among bacteria and eukarya, no homologs have been recognized in the completed genome sequences of any archaea. In this study, MAT has been purified to homogeneity from the archaeon Methanococcus jannaschii, and the gene encoding it has been identified by mass spectrometry. The peptide mass map identifies the gene encoding MAT as MJ1208, a hypothetical open reading frame. The gene was cloned in Escherichia coli, and expressed enzyme has been purified and characterized. This protein has only 22 and 23% sequence identity to the E. coli and human enzymes, respectively, whereas those are 59% identical to each other. The few identical residues include the majority of those constituting the polar active site residues. Each complete archaeal genome sequence contains a homolog of this archaeal-type MAT. Surprisingly, three bacterial genomes encode both the archaeal and eukaryal/bacterial types of MAT. This identification of a second major class of MAT emphasizes the long evolutionary history of the archaeal lineage and the structural diversity found even in crucial metabolic enzymes.

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

Quality and governance in hospitals.

What is the role of the board in quality initiatives in an organization? How can the board improve its own processes through quality initiatives? What are the quality attributes that a board should monitor? The board at St. Mary's Hospital in Kitchener addressed these questions, resulting in a rethinking of the board's role and its relationship to the operation of the hospital. This article discusses how the board has been restructured, and how it has shifted over the past few years to a CQI focus.

Profound ambulatory hypoglycemia: a rare entity.

A 49-year-old man with a history of hepatocellular carcinoma, alcohol abuse, and insulin-dependent diabetes mellitus was noted to be completely asymptomatic despite a plasma glucose level of 4 mg/dL. The possible pathophysiology of this unusual occurrence of "hypoglycemia unawareness" is discussed.

Exposure of humans to a volatile organic mixture. III. Inflammatory response.

A set of symptoms has been described during the past two decades that has been called the "sick building syndrome." These symptoms include eye, nose, and throat irritation; headache; mental fatigue; and respiratory distress. It is likely that the volatile organic compounds (VOCs) present in synthetic materials used in homes and office buildings contribute to these symptoms. However, there have been very few studies in which humans have been exposed to known amounts of VOCs under carefully controlled conditions. In this study, 14 subjects were exposed to a mixture of VOCs (25 mg/m3 total hydrocarbon) that is representative of what is found in new homes and office buildings. Because irritations of the nose and throat are symptoms often associated with the upper respiratory tract and may result from an inflammatory response in the upper airways, we used nasal lavage to monitor neutrophil (PMN) influx into the nasal passages following exposure to VOCs. There were statistically significant increases in PMNs, both immediately after a 4-h exposure to VOCs and 18 h later.

Biomarkers of inflammation in ozone-exposed humans. Comparison of the nasal and bronchoalveolar lavage.

Previously we established that an acute inflammatory response in the upper respiratory tract of humans could be studied by analyses of nasal lavages (NL). The relationship of these cellular responses to responses in the lower lung has not been thoroughly investigated in humans. In this study we have compared the cellular changes detected in NL with those detected in the bronchoalveolar lavage (BAL) taken from the same individual. A group of 10 subjects was exposed to either filtered air or 0.4 ppm ozone (O3), with exercise, for 2 h. The NL was done prior to, immediately following, and 18 h postexposure; the BAL was done only at 18 h postexposure. A significant increase in PMN was detected in the NL immediately postexposure to O3 (7.7-fold increase; p = 0.003) and remained elevated in the 18 h post-O3 NL (6.1-fold increase; p less than 0.001). A similar increase in PMN was detected in the BAL 18 h after exposure to O3 (6.0-fold increase; p less than 0.001). The albumin levels in the NL and BAL were also similarly increased 18 h after O3 (3.9-fold and 2.2-fold, respectively). Although a qualitative correlation in the mean number of PMN existed between the upper and lower respiratory tract after O3, comparison of the NL and BAL PMN from each individual showed a significant quantitative correlation for the air data (r = 0.741; p = 0.014) but not for the O3 data (r = 0.408; p = 0.243). This study demonstrates that PMN counts in the NL can be a useful, inexpensive means of studying the acute inflammatory effect of ozone and monitoring those effects in the lower lung.

Nasal lavage as a tool in assessing acute inflammation in response to inhaled pollutants.

The upper airway, especially the nose, is a major target of toxic damage. Nasal challenges followed by nasal lavage (NAL) have been applied to studies of hypersensitivity, in particular as a method to identify the allergen in patients with allergic situations such as rhinitis. The NAL method has not been extensively used to determine the effects of air pollutants on the upper airways in humans. Ozone is known to interact avidly with various tissues in the respiratory tract and to cause decrements in lung function tests. This oxidant pollutant has also been shown to induce inflammation in the lower airways of humans and animals. In this study, we have examined the effect of an acute (2 h) exposure of ozone at 0.4 ppm on the inflammatory response in the upper airways of 10 normal volunteers and compared these results to those obtained in the lower airways assessed by bronchoalveolar lavage (BAL). The results indicate significant increases in the number of polymorphonuclear neutrophils (PMN) in NAL immediately post exposure (7.7-fold). This increase is still detectable 18 h post exposure (6-fold) which is similar to the increase of PMN in BAL. Tryptase, released by mast cells was also increased in the NAL fluid immediately post exposure (2-fold). While the albumin level, which is an indicator of epithelial cell permeability, was elevated 18 h post exposure (1.5-fold), tryptase level, was not anymore elevated at that time point. Interestingly, several other markers of acute inflammation such as prostaglandin E2 (PGE2), C3a, urokinase-type plasminogen activator (U-PA), which were found to be significantly elevated in the BAL of the same group of subjects (18 h post exposure), were not elevated in the NAL either immediately post or 18 h post exposure. The level of uric acid, thought to be an important anti-oxidant molecule, was also unchanged in the NAL fluid but was elevated in the BAL fluid. Collectively the data suggest that NAL may serve as a sensitive and reliable technique to detect inflammation in the upper airways of subjects exposed to ozone. Moreover, in the case of this particular oxidant pollutant, the NAL seems to mirror the inflammatory response in the lower airways, 18 h post exposure, relative to the number of PMN and albumin levels.

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

Rat C6 glial cells synthesize insulin-like growth factor I (IGF-I) and express IGF-I receptors and IGF-II/mannose 6-phosphate receptors.

We have used the rat C6 glial cell line as a model system to study the role of insulin-like growth factors (IGF) in neuroglial cells of the central nervous system (CNS). Northern blot analysis of C6 RNA demonstrated the presence of IGF-I mRNA and undetectable IGF-II mRNA. IGF-I and IGF-binding protein(s), but not IGF-II, were detected in C6 glial cell-conditioned medium. The level of IGF-I was 1-4 ng/ml in conditioned medium based on a human IGF-I standard. The immunoreactive IGF-I inhibited [125I]IGF-I binding to the IGF-I receptor on chick embryo fibroblasts and stimulated [3H]thymidine incorporation into chick embryo fibroblast DNA. Competitive binding and affinity cross-linking experiments using [125]IGF-I and [125I]IGF-II demonstrated the presence of IGF-I receptors (type I) and IGF-II/mannose 6-phosphate receptors (type II) on C6 glial cell membranes. An immunoglobulin (no. 3637) directed against the rat IGF-II receptor blocked the degradation of [125I]IGF-II added to C6 glial cells, presumably by blocking receptor-mediated internalization. We were unable to demonstrate an autocrine role for IGF in the C6 glial cell line, since [3H]thymidine incorporation into DNA was stimulated equally well by IGF-I-deficient rat serum and normal serum, and added IGF did not stimulate [3H]thymidine incorporation into DNA when tested alone or when added to IGF-I-deficient serum. We propose that neuroglial cell-derived IGF-I may serve as a paracrine growth stimulus in the central nervous system.

Ozone-induced inflammation in the lower airways of human subjects.

Although ozone (O3) has been shown to induce inflammation in the lungs of animals, very little is known about its inflammatory effects on humans. In this study, 11 healthy nonsmoking men, 18 to 35 yr of age (mean, 25.4 +/- 3.5), were exposed once to 0.4 ppm O3 and once to filtered air for 2 h with intermittent exercise. Eighteen hours later, bronchoalveolar lavage (BAL) was performed and the cells and fluid were analyzed for various indicators of inflammation. There was an 8.2-fold increase in the percentage of polymorphonuclear leukocytes (PMN) in the total cell population, and a small but significant decrease in the percentage of macrophages after exposure to O3. Immunoreactive neutrophil elastase often associated with inflammation and lung damage increased by 3.8-fold in the fluid while its activity increased 20.6-fold in the lavaged cells. A 2-fold increase in the levels of protein, albumin, and IgG suggested increased vascular permeability of the lung. Several biochemical markers that could act as chemotactic or regulatory factors in an inflammatory response were examined in the BAL fluid (BALF). The level of complement fragment C3 alpha was increased by 1.7-fold. The chemotactic leukotriene B4 was unchanged while prostaglandin E2 increased 2-fold. In contrast, three enzyme systems of phagocytes with potentially damaging effects on tissues and microbes, namely, NADPH-oxidase and the lysosomal enzymes acid phosphatase and beta-glucuronidase, were increased neither in the lavaged fluid nor cells. In addition, the amounts of fibrogenic-related molecules were assessed in BALF.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and expression of the rat insulin-like growth factor II (rIGF-II) gene. rIGF-II RNAs are transcribed from two promoters.

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5'-noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age.

Developmental regulation of insulin-like growth factor II mRNA in different rat tissues.

Insulin-like growth factor II (IGF-II) is present at high levels in fetal and early neonatal rat plasma, and decreases profoundly following birth. In the present study, the levels of IGF-II RNA in different rat tissues at different ages were determined by hybridization to a rat IGF-II cDNA probe. IGF-II RNA was present in 11 of 13 fetal or neonatal tissues examined: at higher levels in muscle, skin, lung, liver, intestine, and thymus; at lower levels in brain stem, heart, cerebral cortex, kidney, and hypothalamus; and undetectable in spleen and pancreas (although the latter RNA was partially degraded). In each tissue, Northern blot hybridization revealed the presence of six IGF-II RNAs: 6, 4, 3.8, 2.2, 1.7, and 1.2 kilobase pairs, consistent with results previously observed in the BRL-3A rat liver cell line and attributed to alternative RNA processing. Although differences in the relative abundance of these RNAs were observed in different tissues, the same size species occurred in all tissues with the 4-kilobase pair RNA the most abundant species. RNAs from the different tissues were examined at six developmental ages (days 16 and 21 of gestation; days 2, 11, 22, and 75 after birth) by hybridization to slot blots and Northern blots. In lung, thymus, kidney, and brain stem, IGF-II RNA was expressed at higher levels in the fetus than after birth, whereas in muscle, skin, liver, heart, and intestine, the high fetal levels of IGF-II RNA continued through day 11 or day 22 after birth. IGF-II RNA persisted into adulthood in cerebral cortex and hypothalamus. Although the significance of these tissue-specific differences in the developmental regulation of the expression of IGF-II RNA remains to be established, they exhibit intriguing temporal correlations with major maturational events in some tissues such as lung and muscle.