Complete Genome Sequence of Alkaliphilus metalliredigens Strain QYMF, an Alkaliphilic and Metal-Reducing Bacterium Isolated from Borax-Contaminated Leachate Ponds.

Alkaliphilus metalliredigens strain QYMF is an anaerobic, alkaliphilic, and metal-reducing bacterium associated with phylum Firmicutes QYMF was isolated from alkaline borax leachate ponds. The genome sequence will help elucidate the role of metal-reducing microorganisms under alkaline environments, a capability that is not commonly observed in metal respiring-microorganisms.

Complete Genome Sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment.

We report the genome sequence of Anaeromyxobacter sp. Fw109-5, isolated from nitrate- and uranium-contaminated subsurface sediment of the Oak Ridge Integrated Field-Scale Subsurface Research Challenge (IFC) site, Oak Ridge Reservation, TN. The bacterium's genome sequence will elucidate its physiological potential in subsurface sediments undergoing in situ uranium bioremediation and natural attenuation.

The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

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.

Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.

The genome of a motile marine Synechococcus.

Marine unicellular cyanobacteria are responsible for an estimated 20-40% of chlorophyll biomass and carbon fixation in the oceans. Here we have sequenced and analysed the 2.4-megabase genome of Synechococcus sp. strain WH8102, revealing some of the ways that these organisms have adapted to their largely oligotrophic environment. WH8102 uses organic nitrogen and phosphorus sources and more sodium-dependent transporters than a model freshwater cyanobacterium. Furthermore, it seems to have adopted strategies for conserving limited iron stores by using nickel and cobalt in some enzymes, has reduced its regulatory machinery (consistent with the fact that the open ocean constitutes a far more constant and buffered environment than fresh water), and has evolved a unique type of swimming motility. The genome of WH8102 seems to have been greatly influenced by horizontal gene transfer, partially through phages. The genetic material contributed by horizontal gene transfer includes genes involved in the modification of the cell surface and in swimming motility. On the basis of its genome, WH8102 is more of a generalist than two related marine cyanobacteria.

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L.

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

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.

The photosynthetic apparatus of Prochlorococcus: Insights through comparative genomics.

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.

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

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.

D-ribose-5-phosphate isomerase from spinach: heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme.

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.

Protein threading by PROSPECT: a prediction experiment in CASP3.

We present an analysis of the protein fold recognition experiment using PROSPECT in The Third Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP3). PROSPECT is a computer program we have recently developed for finding an optimal alignment between a protein sequence and a protein structural fold. Two unique features of PROSPECT are (a) that it guarantees to find the globally optimal sequence-structure alignment and does so in an efficient manner, when the alignment-scoring function consists of three additive terms: (i) a singleton fitness term, (ii) a pairwise contact preference term between residues that are spatially close (

Identification of a catalytic aspartyl residue of D-ribulose 5-phosphate 3-epimerase by site-directed mutagenesis.

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs.

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.

D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme.

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Roles and microenvironments of tryptophanyl residues of spinach phosphoribulokinase.

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.

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.

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.

Mapping individual cosmid DNAs by direct AFM imaging.

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.

Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.

The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77. Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared. All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax. In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins. The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f. Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.