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Halorubrum lacusprofundi is an extreme halophile within the archaeal phylum Euryarchaeota. The type strain ACAM 34 was isolated from Deep Lake, Antarctica. H. lacusprofundi is of phylogenetic interest because it is distantly related to the haloarchaea that have previously been sequenced. It is also of interest because of its psychrotolerance. We report here the complete genome sequence of H. lacusprofundi type strain ACAM 34 and its annotation. This genome is part of a 2006 Joint Genome Institute Community Sequencing Program project to sequence genomes of diverse Archaea.

Desulfotomaculum ruminis Campbell and Postgate 1965 is a member of the large genus Desulfotomaculum which contains 30 species and is contained in the family Peptococcaceae. This species is of interest because it represents one of the few sulfate-reducing bacteria that have been isolated from the rumen. Here we describe the features of D. ruminis together with the complete genome sequence and annotation. The 3,969,014 bp long chromosome with a total of 3,901 protein-coding and 85 RNA genes is the second completed genome sequence of a type strain of the genus Desulfotomaculum to be published, and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2009.

Thauera aminoaromatica strain MZ1T, an isolate belonging to genus Thauera, of the family Rhodocyclaceae and the class the Betaproteobacteria, has been characterized for its ability to produce abundant exopolysaccharide and degrade various aromatic compounds with nitrate as an electron acceptor. These properties, if fully understood at the genome-sequence level, can aid in environmental processing of organic matter in anaerobic cycles by short-circuiting a central anaerobic metabolite, acetate, from microbiological conversion to methane, a critical greenhouse gas. Strain MZ1T is the first strain from the genus Thauera with a completely sequenced genome. The 4,496,212 bp chromosome and 78,374 bp plasmid contain 4,071 protein-coding and 71 RNA genes, and were sequenced as part of the DOE Community Sequencing Program CSP_776774.

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.

The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacteria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. baltica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacterium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 protein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008.

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called 'moderately halophilic bacteria' currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11(T) and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004.

Tolumonas auensis Fischer-Romero et al. 1996 is currently the only validly named species of the genus Tolumonas in the family Aeromonadaceae. The strain is of interest because of its ability to produce toluene from phenylalanine and other phenyl precursors, as well as phenol from tyrosine. This is of interest because toluene is normally considered to be a tracer of anthropogenic pollution in lakes, but T. auensis represents a biogenic source of toluene. Other than Aeromonas hydrophila subsp. hydrophila, T. auensis strain TA 4(T) is the only other member in the family Aeromonadaceae with a completely sequenced type-strain genome. The 3,471,292 bp chromosome with a total of 3,288 protein-coding and 116 RNA genes was sequenced as part of the DOE Joint Genome Institute Program JBEI 2008.

Ferroglobus placidus belongs to the order Archaeoglobales within the archaeal phylum Euryarchaeota. Strain AEDII12DO is the type strain of the species and was isolated from a shallow marine hydrothermal system at Vulcano, Italy. It is a hyperthermophilic, anaerobic chemolithoautotroph, but it can also use a variety of aromatic compounds as electron donors. Here we describe the features of this organism together with the complete genome sequence and annotation. The 2,196,266 bp genome with its 2,567 protein-coding and 55 RNA genes was sequenced as part of a DOE Joint Genome Institute Laboratory Sequencing Program (LSP) project.

Desulfovibrio alaskensis G20 (formerly Desulfovibrio desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7-Mb genome sequence to provide insights into its physiology.

Rhodospirillum rubrum (Esmarch 1887) Molisch 1907 is the type species of the genus Rhodospirillum, which is the type genus of the family Rhodospirillaceae in the class Alphaproteobacteria. The species is of special interest because it is an anoxygenic phototroph that produces extracellular elemental sulfur (instead of oxygen) while harvesting light. It contains one of the most simple photosynthetic systems currently known, lacking light harvesting complex 2. Strain S1(T) can grow on carbon monoxide as sole energy source. With currently over 1,750 PubMed entries, R. rubrum is one of the most intensively studied microbial species, in particular for physiological and genetic studies. Next to R. centenum strain SW, the genome sequence of strain S1(T) is only the second genome of a member of the genus Rhodospirillum to be published, but the first type strain genome from the genus. The 4,352,825 bp long chromosome and 53,732 bp plasmid with a total of 3,850 protein-coding and 83 RNA genes were sequenced as part of the DOE Joint Genome Institute Program DOEM 2002.

Here we present the genome of strain Exiguobacterium sp. AT1b, a thermophilic member of the genus Exiguobacterium whose representatives were isolated from various environments along a thermal and physicochemical gradient. This genome was sequenced to be a comparative resource for the study of thermal adaptation with a psychroactive representative of the genus, Exiguobacterium sibiricum strain 255-15, that was previously sequenced by the U.S. Department of Energy's (DOE's) Joint Genome Institute (JGI) (http://genome.ornl.gov/microbial/exig/).

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as -10 degrees C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (-10 degrees C to -12 degrees C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment., Competing Interests: The authors have declared that no competing interests exist.

Unlike the classical Yersinia pestis strains, members of an atypical group of Y. pestis from Central Asia, denominated Y. pestis subspecies caucasica (also known as one of several pestoides types), are distinguished by a number of characteristics including their ability to ferment rhamnose and melibiose, their lack of the small plasmid encoding the plasminogen activator (pla) and pesticin, and their exceptionally large variants of the virulence plasmid pMT (encoding murine toxin and capsular antigen). We have obtained the entire genome sequence of Y. pestis Pestoides F, an isolate from the former Soviet Union that has enabled us to carryout a comprehensive genome-wide comparison of this organism's genomic content against the six published sequences of Y. pestis and their Y. pseudotuberculosis ancestor. Based on classical glycerol fermentation (+ve) and nitrate reduction (+ve) Y. pestis Pestoides F is an isolate that belongs to the biovar antiqua. This strain is unusual in other characteristics such as the fact that it carries a non-consensus V antigen (lcrV) sequence, and that unlike other Pla(-) strains, Pestoides F retains virulence by the parenteral and aerosol routes. The chromosome of Pestoides F is 4,517,345 bp in size comprising some 3,936 predicted coding sequences, while its pCD and pMT plasmids are 71,507 bp and 137,010 bp in size respectively. Comparison of chromosome-associated genes in Pestoides F with those in the other sequenced Y. pestis strains reveals differences ranging from strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. There is a single approximately 7 kb unique region in the chromosome not found in any of the completed Y. pestis strains sequenced to date, but which is present in the Y. pseudotuberculosis ancestor. Taken together, these findings are consistent with Pestoides F being derived from the most ancient lineage of Y. pestis yet sequenced.

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the "classical" antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. We compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.

The complete genomic sequence of Pseudomonas syringae pv. syringae B728a (Pss B728a) has been determined and is compared with that of P. syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 Mb) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. Although a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared with Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences unique to Pss B728a when compared with Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands varies, with one containing a prophage and another the plasmid pKLC102 of Pseudomonas aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin, syringomycin, indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to the epiphytic fitness and stress tolerance of this organism.

We analyzed the amino acid composition of different categories of proteins of the moderately halophilic bacterium Chromohalobacter salexigens, as deduced from its genome sequence. Comparison with non-halophilic representatives of the gamma-Proteobacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae) shows only a slight excess of acidic residues in the cytoplasmic proteins, and no significant differences were found in the acidity of membrane-bound proteins. In contrast, a very pronounced difference in mean pI value was observed for the periplasmic binding proteins of the ABC transport systems of C. salexigens and the non-halophiles E. coli and P. aeruginosa. V. cholerae, which is adapted to life in brackish water, showed intermediate values. The findings suggest that there is a major difference between the proteins of the moderate halophile C. salexigens and non-halophilic bacteria in their periplasmic proteins, exemplified by the substrate binding proteins of transport systems. The highly acidic nature of these proteins may enable them to function at high salt concentrations. The evolution of highly salt-tolerant prokaryotes may have depended on an increase in acidity of the proteins located external to the cytoplasmic membrane, enabling effective transport of nutrients into the cell.

White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15 degrees -98 degrees C) were used to generate 1111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gln, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60 degrees C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.

The genomes of many organisms have been sequenced in the last 5 years. Typically about 30% of predicted genes from a newly sequenced genome cannot be given functional assignments using sequence comparison methods. In these situations three-dimensional structural predictions combined with a suite of computational tools can suggest possible functions for these hypothetical proteins. Suggesting functions may allow better interpretation of experimental data (e.g., microarray data and mass spectroscopy data) and help experimentalists design new experiments. In this paper, we focus on three hypothetical proteins of Shewanella oneidensis MR-1 that are potentially related to iron transport/metabolism based on microarray experiments. The threading program PROSPECT was used for protein structural predictions and functional annotation, in conjunction with literature search and other computational tools. Computational tools were used to perform transmembrane domain predictions, coiled coil predictions, signal peptide predictions, sub-cellular localization predictions, motif prediction, and operon structure evaluations. Combined computational results from all tools were used to predict roles for the hypothetical proteins. This method, which uses a suite of computational tools that are freely available to academic users, can be used to annotate hypothetical proteins in general.

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with approximately 47% transcribed from one strand and approximately 53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO(2) and NH(3) assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.

Within the vast oceanic gyres, a significant fraction of the total chlorophyll belongs to the light-harvesting antenna systems of a single genus, Prochlorococcus. This organism, discovered only about 10 years ago, is an extremely small, Chl b-containing cyanobacterium that sometimes constitutes up to 50% of the photosynthetic biomass in the oceans. Various Prochlorococcus strains are known to have significantly different conditions for optimal growth and survival. Strains which dominate the surface waters, for example, have an irradiance optimum for photosynthesis of 200 mumol photons m(-2) s(-1), whereas those that dominate the deeper waters photosynthesize optimally at 30-50 mumol photons m(-2) s(-1). These high and low light adapted 'ecotypes' are very closely related - less than 3% divergent in their 16S rRNA sequences - inviting speculation as to what features of their photosynthetic mechanisms might account for the differences in photosynthetic performance. Here, we compare information obtained from the complete genome sequences of two Prochlorococcus strains, with special emphasis on genes for the photosynthetic apparatus. These two strains, Prochlorococcus MED4 and MIT 9313, are representatives of high- and low-light adapted ecotypes, characterized by their low or high Chl b/a ratio, respectively. Both genomes appear to be significantly smaller (1700 and 2400 kbp) than those of other cyanobacteria, and the low-light-adapted strain has significantly more genes than its high light counterpart. In keeping with their comparative light-dependent physiologies, MED4 has many more genes encoding putative high-light-inducible proteins (HLIP) and photolyases to repair UV-induced DNA damage, whereas MIT 9313 possesses more genes associated with the photosynthetic apparatus. These include two pcb genes encoding Chl-binding proteins and a second copy of the gene psbA, encoding the Photosystem II reaction center protein D1. In addition, MIT 9313 contains a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate form between the phycobiliproteins of non-Chl b containing cyanobacteria and an extremely modified beta phycoerythrin as the sole derivative of phycobiliproteins still present in MED4. Intriguing features found in both Prochlorococcus strains include a gene cluster for Rubisco and carboxysomal proteins that is likely of non-cyanobacterial origin and two genes for a putative varepsilon and beta lycopene cyclase, respectively, explaining how Prochlorococcus may synthesize the alpha branch of carotenoids that are common in green organisms but not in other cyanobacteria.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178-184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a K(i) of 7.9 mM. D-Arabinose 5-phosphate is an effective inhibitor, while D-xylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although D-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. D87A, K100A, and D90A mutants show greatly diminished k(cat) values (0. 0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in K(m) of D-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues., (Copyright 2000 Academic Press.)

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Phosphoribulokinase is one of several Calvin cycle enzymes that are light-regulated via the ferredoxin-thioredoxin system (R. A. Wolosiuk and B. B. Buchanan, 1978, Arch. Biochem. Biophys. 189, 97-101). Substitution of the only two Trp residues of the enzyme was prompted by the following goals: to identify each tryptophanyl residue with respect to prior classifications as exposed and buried (C. A. Ghiron et al., 1988, Arch. Biochem. Biophys. 260, 267-272); to explore the possible active-site location and function of conserved Trp155, as suggested by sequence proximity to catalytic Asp160 (H. A. Charlier et al., 1994, Biochemistry 33, 9343-9350); and to determine if fluorescence of a Trp residue can serve as a gauge of conformational differences between the reduced (active) and the oxidized (inactive) forms of the enzyme. Emission spectra and acrylamide quenching data demonstrate that Trp155 is solvent exposed, while Trp241 is buried. Kinetic parameters of the W241F mutant are not significantly altered relative to those of wild-type enzyme, thereby discounting any requirement for Trp at position 241. While substitution of Trp155 with Phe or Ala has little impact on Vmax, the Km for Ru5P and ATP are increased substantially; the diminished affinity for ATP is particularly pronounced in the case of the Ala substitution. In further support of an active-site location of Trp155, its fluorescence emission is subject to quenching by nucleotides. Fluorescence quenching of reduced W241F by ATP gives a dissociation constant (Kd) of 37 microM, virtually identical with its Km of 46 microM, and provides for the first time a direct measurement of the interaction of the kinase with product ADP (Kd of 1.3 mM). Fluorescence quenching of oxidized W241F by ATP reveals a Kd of 28 mM; however, this weakened binding does not reflect an altered microenvironment of Trp155, as its steady-state emission and fluorescence lifetimes are unaffected by the oxidation state., (Copyright 1998 Academic Press.)

Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.

Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

Frameshift mutations occur by a number of mechanisms. To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae. All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38. DNA sequence information was recovered using two approaches-the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR). Using these techniques, saturated mutation spectra for the spontaneous reversion of his4-38 were generated. The most frequently occurring mutational events in both strains were -1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed. Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of -1 frameshift mutations. In addition, while for -1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only -1 frameshifts and one +2 frameshift), and those recovered using PCR (-1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations). This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations.

To evaluate the functions of flexible loop 6 at the active site of Rhodospirillum rubrum D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the loop was truncated by cassette mutagenesis, whereby seven residues of the twelve-residue loop were excised and replaced by two glycyl residues. The purified loop-deletion mutant was totally devoid of carboxylase activity, but retained substantial catalytic competency in the enolization of ribulose bisphosphate (the initial step in the overall carboxylase pathway) and in normal processing of the six-carbon carboxylated intermediate (the terminal steps in the overall carboxylase pathway). Hence, catalytic impairment resides predominantly at the stage of carboxylation of the initial enediol(ate), a conclusion compatible with mechanistic deductions derived from crystallographic analyses. A critical role of loop 6 in the stabilization of the transition state for carboxylation is reinforced by the findings that the loop-deletion mutant displays preferentially compromised affinity for an analogue of the carboxylated intermediate relative to ribulose bisphosphate and that the mutant converts the substrate to a dicarbonyl compound as a consequence of beta-elimination of phosphate from the initial enediol(ate).

Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.

As a prerequisite to protein engineering, we have overexpressed the rbcLS operon of the cyanobacterium Anabaena 7120, in Escherichia coli. The operon encodes the large and small subunits of ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Levels of active enzyme exceed 6% of soluble protein. We noted an apparent third gene, an unidentified open reading frame (URF) referred to here as rbcX, in the 558-bp intergenic space between the large and small subunit encoding genes. The URF, rbcX, has no known function. High-level production of Rubisco activity from the rbc operon in E. coli required simultaneous overproduction of the GroESL chaperonins under a regimen of limited growth, in contrast to more modest conditions which suffice for efficient production of the Anacystis nidulans cyanobacterial Rubisco. Deletion of rbcX or inversion of the rbcL-rbcS order did not enhance expression levels. The recombinant Rubisco, purified to near homogeneity, exhibited functional properties [Km(ribulose-P2), kcat, transition-state-analogue binding stoichiometry/exchange, and specificity factor] essentially identical to those of the enzyme obtained from Anabaena.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.

Crystallographic studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum suggest that active-site Asn111 interacts with Mg2+ and/or substrate (Lundqvist, T., and Schneider, G. (1991) J. Biol. Chem. 266, 12604-12611). To examine possible catalytic roles of Asn111, we have used site-directed mutagenesis to replace it with a glutaminyl, aspartyl, seryl, or lysyl residue. Although the mutant proteins are devoid of detectable carboxylase activity, their ability to form a quaternary complex comprised of CO2, Mg2+, and a reaction-intermediate analogue is indicative of competence in activation chemistry and substrate binding. The mutant proteins retain enolization activity, as measured by exchange of the C3 proton of ribulose bisphosphate with solvent, thereby demonstrating a preferential role of Asn111 in some later step of overall catalysis. The active sites of this homodimeric enzyme are formed by interactive domains from adjacent subunits (Larimer, F. W., Lee, E. H., Mural, R. J., Soper, T. S., and Hartman, F. C. (1987) J. Biol. Chem. 262, 15327-15329). Crystallography assigns Asn111 to the amino-terminal domain of the active site (Knight, S., Anderson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). The observed formation of enzymatically active heterodimers by the in vivo hybridization of an inactive position-111 mutant with inactive carboxyl-terminal domain mutants is consistent with this assignment.

A mutation spectrum was constructed from a series of randomly isolated spontaneous His+ revertants of the frameshift mutant his4-38 in Saccharomyces cerevisiae. For each true revertant, a 438 bp region encompassing his4-38 on chromosome III was recovered into a shuttle vector by double-strand gap repair. Of the 45 independent His+ revertants sequenced, 44 were -1 base deletions and one revertant was a +2 base insertion. The -1 deletions exhibited a bimodal distribution. Of the bases encompassing the his4-38 region from +153-181, approximately 45% were not involved in a reversion event, although a -1 frameshift within this region will result in a viable His+ revertant. Approximately 49% of -1 events occurred within runs of 3 repeated bases. At these sites the strand-slippage model for frameshift mutation is supported. However, the -1 events occurring at sites of 2 repeated bases and the low frequency (2%) of +2 base insertions suggest that the transiently misaligned template model is a significant mechanism in reversion of his4-38. When the distribution of -1 events at repeated bases was discounted, a hotspot involving a -T at position +163 was resolved.

Numerous candidates have been suggested according to chemical and structural criteria for the active site base of ribulose bisphosphate carboxylase/oxygenase that catalyzes substrate enolization. We evaluate the functional significance of two such candidates, His-321 and Ser-368 of the Rhodospirillum rubrum enzyme, by site-directed mutagenesis. Position 321 mutants retain 3-12% of wild-type rates of both overall carboxylation and the initial enolization, with little effect on Km for CO2 or ribulose bisphosphate. Position 368 mutants exhibit approximately 1% of wild-type carboxylation but 4-9% of enolization, also accompanied by little effect on Km values. The modest catalytic facilitations elicited by these residues are incompatible with either acting as the crucial base. The enhanced efficiency of the position 368 mutants in enolization versus carboxylation clearly indicates that Ser-368 effects catalysis preferentially beyond the point of proton abstraction. Both sets of mutants bind the reaction intermediate analogue, 2-carboxy-D-arabinitol bisphosphate, stoichiometrically. Ligand exchange from complexes with position 321 mutants is increased relative to wild type, whereas complexes with position 368 mutants are more exchange-inert. Therefore, His-321 may assist stabilization of the transition state mimicked by the analogue.

In a scanning tunneling microscope (STM) electrochemical cell we have studied the effects of electrode potential on both the surface topography and the adsorption of deoxyribonucleic acid (DNA) to graphite and gold surfaces. Images of the surface of highly oriented pyrolytic graphite (HOPG), of the same area, in response to a positive increase in surface potential show degradation of the step edges with little change in the crystal plane. Images of the same area of a gold surface demonstrate the formation of and the progressive increase in nodular structures on the crystal planes, in response to increased potential, with little effect on the step edges. Using radio-labeled DNA we monitored electrochemical absorption onto HOPG and gold surfaces. Although at no applied potential and at negative surface potentials some DNA was bound, at positive potentials 3 to 5 times more DNA was incorporated onto both surfaces. DNA adsorbed to a surface at a positive potential was not removed by reversing the potential.