86 results on '"Larimer F"'
Search Results
2. Complete genome sequence of the Antarctic Halorubrum lacusprofundi type strain ACAM 34.
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Anderson IJ, DasSarma P, Lucas S, Copeland A, Lapidus A, Del Rio TG, Tice H, Dalin E, Bruce DC, Goodwin L, Pitluck S, Sims D, Brettin TS, Detter JC, Han CS, Larimer F, Hauser L, Land M, Ivanova N, Richardson P, Cavicchioli R, DasSarma S, Woese CR, and Kyrpides NC
- Abstract
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.
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- 2016
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3. Complete Genome Sequence of Anaeromyxobacter sp. Fw109-5, an Anaerobic, Metal-Reducing Bacterium Isolated from a Contaminated Subsurface Environment.
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Hwang C, Copeland A, Lucas S, Lapidus A, Barry K, Glavina Del Rio T, Dalin E, Tice H, Pitluck S, Sims D, Brettin T, Bruce DC, Detter JC, Han CS, Schmutz J, Larimer FW, Land ML, Hauser LJ, Kyrpides N, Lykidis A, Richardson P, Belieav A, Sanford RA, Löeffler FE, and Fields MW
- Abstract
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., (Copyright © 2015 Hwang et al.)
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- 2015
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4. Complete genome sequence of the sulfate-reducing firmicute Desulfotomaculum ruminis type strain (DL(T)).
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Spring S, Visser M, Lu M, Copeland A, Lapidus A, Lucas S, Cheng JF, Han C, Tapia R, Goodwin LA, Pitluck S, Ivanova N, Land M, Hauser L, Larimer F, Rohde M, Göker M, Detter JC, Kyrpides NC, Woyke T, Schaap PJ, Plugge CM, Muyzer G, Kuever J, Pereira IA, Parshina SN, Bernier-Latmani R, Stams AJ, and Klenk HP
- Abstract
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.
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- 2012
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5. Complete genome sequence of Thauera aminoaromatica strain MZ1T.
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Jiang K, Sanseverino J, Chauhan A, Lucas S, Copeland A, Lapidus A, Del Rio TG, Dalin E, Tice H, Bruce D, Goodwin L, Pitluck S, Sims D, Brettin T, Detter JC, Han C, Chang YJ, Larimer F, Land M, Hauser L, Kyrpides NC, Mikhailova N, Moser S, Jegier P, Close D, Debruyn JM, Wang Y, Layton AC, Allen MS, and Sayler GS
- Abstract
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.
- Published
- 2012
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6. Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T)).
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Meincke L, Copeland A, Lapidus A, Lucas S, Berry KW, Del Rio TG, Hammon N, Dalin E, Tice H, Pitluck S, Richardson P, Bruce D, Goodwin L, Han C, Tapia R, Detter JC, Schmutz J, Brettin T, Larimer F, Land M, Hauser L, Kyrpides NC, Ivanova N, Göker M, Woyke T, Wu QL, Pöckl M, Hahn MW, and Klenk HP
- Abstract
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.
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- 2012
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7. Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11(T)).
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Copeland A, O'Connor K, Lucas S, Lapidus A, Berry KW, Detter JC, Del Rio TG, Hammon N, Dalin E, Tice H, Pitluck S, Bruce D, Goodwin L, Han C, Tapia R, Saunders E, Schmutz J, Brettin T, Larimer F, Land M, Hauser L, Vargas C, Nieto JJ, Kyrpides NC, Ivanova N, Göker M, Klenk HP, Csonka LN, and Woyke T
- Abstract
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.
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- 2011
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8. Complete genome sequence of Hirschia baltica type strain (IFAM 1418(T)).
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Chertkov O, Brown PJ, Kysela DT, de Pedro MA, Lucas S, Copeland A, Lapidus A, Del Rio TG, Tice H, Bruce D, Goodwin L, Pitluck S, Detter JC, Han C, Larimer F, Chang YJ, Jeffries CD, Land M, Hauser L, Kyrpides NC, Ivanova N, Ovchinnikova G, Tindall BJ, Göker M, Klenk HP, and Brun YV
- Abstract
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.
- Published
- 2011
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9. Complete genome sequence of Ferroglobus placidus AEDII12DO.
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Anderson I, Risso C, Holmes D, Lucas S, Copeland A, Lapidus A, Cheng JF, Bruce D, Goodwin L, Pitluck S, Saunders E, Brettin T, Detter JC, Han C, Tapia R, Larimer F, Land M, Hauser L, Woyke T, Lovley D, Kyrpides N, and Ivanova N
- Abstract
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.
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- 2011
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10. Complete genome sequence of Tolumonas auensis type strain (TA 4).
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Chertkov O, Copeland A, Lucas S, Lapidus A, Berry KW, Detter JC, Del Rio TG, Hammon N, Dalin E, Tice H, Pitluck S, Richardson P, Bruce D, Goodwin L, Han C, Tapia R, Saunders E, Schmutz J, Brettin T, Larimer F, Land M, Hauser L, Spring S, Rohde M, Kyrpides NC, Ivanova N, Göker M, Beller HR, Klenk HP, and Woyke T
- Abstract
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.
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- 2011
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11. Complete genome sequence and updated annotation of Desulfovibrio alaskensis G20.
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Hauser LJ, Land ML, Brown SD, Larimer F, Keller KL, Rapp-Giles BJ, Price MN, Lin M, Bruce DC, Detter JC, Tapia R, Han CS, Goodwin LA, Cheng JF, Pitluck S, Copeland A, Lucas S, Nolan M, Lapidus AL, Palumbo AV, and Wall JD
- Subjects
- Base Sequence, Desulfovibrio physiology, Molecular Sequence Data, Desulfovibrio classification, Desulfovibrio genetics, Genome, Bacterial
- Abstract
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.
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- 2011
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12. Complete genome sequence of Rhodospirillum rubrum type strain (S1).
- Author
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Munk AC, Copeland A, Lucas S, Lapidus A, Del Rio TG, Barry K, Detter JC, Hammon N, Israni S, Pitluck S, Brettin T, Bruce D, Han C, Tapia R, Gilna P, Schmutz J, Larimer F, Land M, Kyrpides NC, Mavromatis K, Richardson P, Rohde M, Göker M, Klenk HP, Zhang Y, Roberts GP, Reslewic S, and Schwartz DC
- Abstract
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.
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- 2011
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13. Complete genome sequence of the Thermophilic Bacterium Exiguobacterium sp. AT1b.
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Vishnivetskaya TA, Lucas S, Copeland A, Lapidus A, Glavina del Rio T, Dalin E, Tice H, Bruce DC, Goodwin LA, Pitluck S, Saunders E, Brettin T, Detter C, Han C, Larimer F, Land ML, Hauser LJ, Kyrpides NC, Ovchinnikova G, Kathariou S, Ramaley RF, Rodrigues DF, Hendrix C, Richardson P, and Tiedje JM
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- Bacillales isolation & purification, Environmental Microbiology, Hot Temperature, Molecular Sequence Data, Bacillales genetics, DNA, Bacterial chemistry, DNA, Bacterial genetics, Genome, Bacterial, Sequence Analysis, DNA
- Abstract
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/).
- Published
- 2011
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14. The genome sequence of Psychrobacter arcticus 273-4, a psychroactive Siberian permafrost bacterium, reveals mechanisms for adaptation to low-temperature growth.
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Ayala-del-Río HL, Chain PS, Grzymski JJ, Ponder MA, Ivanova N, Bergholz PW, Di Bartolo G, Hauser L, Land M, Bakermans C, Rodrigues D, Klappenbach J, Zarka D, Larimer F, Richardson P, Murray A, Thomashow M, and Tiedje JM
- Subjects
- Cold Temperature, Freezing, Molecular Sequence Data, Psychrobacter isolation & purification, Psychrobacter physiology, Sequence Analysis, DNA, Siberia, Soil Microbiology, DNA, Bacterial chemistry, DNA, Bacterial genetics, Genome, Bacterial, Psychrobacter genetics
- Abstract
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.
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- 2010
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15. Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T.
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Weiner RM, Taylor LE 2nd, Henrissat B, Hauser L, Land M, Coutinho PM, Rancurel C, Saunders EH, Longmire AG, Zhang H, Bayer EA, Gilbert HJ, Larimer F, Zhulin IB, Ekborg NA, Lamed R, Richardson PM, Borovok I, and Hutcheson S
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- Alteromonadaceae chemistry, Alteromonadaceae enzymology, Alteromonadaceae metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Molecular Sequence Data, Polysaccharides chemistry, Polysaccharides genetics, Protein Transport, Sequence Analysis, DNA, Signal Transduction, Substrate Specificity, Alteromonadaceae genetics, Chromosome Mapping, Genome, Bacterial, Polysaccharides metabolism, Seawater microbiology
- Abstract
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.
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- 2008
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16. Pestoides F, an atypical Yersinia pestis strain from the former Soviet Union.
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Garcia E, Worsham P, Bearden S, Malfatti S, Lang D, Larimer F, Lindler L, and Chain P
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- Amino Acid Sequence, Antigens, Bacterial genetics, Genome, Bacterial, Molecular Sequence Data, Pore Forming Cytotoxic Proteins genetics, Sequence Homology, Amino Acid, Species Specificity, USSR, Virulence genetics, Yersinia pestis genetics, Yersinia pestis metabolism, Yersinia pseudotuberculosis genetics, Yersinia pestis classification, Yersinia pestis isolation & purification
- Abstract
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.
- Published
- 2007
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17. Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.
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Chain PS, Denef VJ, Konstantinidis KT, Vergez LM, Agulló L, Reyes VL, Hauser L, Córdova M, Gómez L, González M, Land M, Lao V, Larimer F, LiPuma JJ, Mahenthiralingam E, Malfatti SA, Marx CJ, Parnell JJ, Ramette A, Richardson P, Seeger M, Smith D, Spilker T, Sul WJ, Tsoi TV, Ulrich LE, Zhulin IB, and Tiedje JM
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- Burkholderia chemistry, Burkholderia metabolism, Burkholderia pathogenicity, Chromosomes, Bacterial, Evolution, Molecular, Gene Expression Profiling, Molecular Structure, Oligonucleotide Array Sequence Analysis, Burkholderia genetics, Genome, Bacterial, Replicon
- Abstract
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.
- Published
- 2006
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18. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).
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Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP, Blaudez D, Boerjan W, Brun A, Brunner A, Busov V, Campbell M, Carlson J, Chalot M, Chapman J, Chen GL, Cooper D, Coutinho PM, Couturier J, Covert S, Cronk Q, Cunningham R, Davis J, Degroeve S, Déjardin A, Depamphilis C, Detter J, Dirks B, Dubchak I, Duplessis S, Ehlting J, Ellis B, Gendler K, Goodstein D, Gribskov M, Grimwood J, Groover A, Gunter L, Hamberger B, Heinze B, Helariutta Y, Henrissat B, Holligan D, Holt R, Huang W, Islam-Faridi N, Jones S, Jones-Rhoades M, Jorgensen R, Joshi C, Kangasjärvi J, Karlsson J, Kelleher C, Kirkpatrick R, Kirst M, Kohler A, Kalluri U, Larimer F, Leebens-Mack J, Leplé JC, Locascio P, Lou Y, Lucas S, Martin F, Montanini B, Napoli C, Nelson DR, Nelson C, Nieminen K, Nilsson O, Pereda V, Peter G, Philippe R, Pilate G, Poliakov A, Razumovskaya J, Richardson P, Rinaldi C, Ritland K, Rouzé P, Ryaboy D, Schmutz J, Schrader J, Segerman B, Shin H, Siddiqui A, Sterky F, Terry A, Tsai CJ, Uberbacher E, Unneberg P, Vahala J, Wall K, Wessler S, Yang G, Yin T, Douglas C, Marra M, Sandberg G, Van de Peer Y, and Rokhsar D
- Subjects
- Arabidopsis genetics, Chromosome Mapping, Computational Biology, Evolution, Molecular, Expressed Sequence Tags, Gene Expression, Genes, Plant, Oligonucleotide Array Sequence Analysis, Phylogeny, Plant Proteins chemistry, Plant Proteins genetics, Polymorphism, Single Nucleotide, Populus growth & development, Populus metabolism, Protein Structure, Tertiary, RNA, Plant analysis, RNA, Untranslated analysis, Gene Duplication, Genome, Plant, Populus genetics, Sequence Analysis, DNA
- Abstract
We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.
- Published
- 2006
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19. Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516: evidence of gene reduction in an emerging pathogen.
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Chain PS, Hu P, Malfatti SA, Radnedge L, Larimer F, Vergez LM, Worsham P, Chu MC, and Andersen GL
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- Bacterial Proteins genetics, Gene Deletion, Molecular Sequence Data, Open Reading Frames, Polymorphism, Single Nucleotide, Species Specificity, Genome, Bacterial, Yersinia pestis genetics
- Abstract
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.
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- 2006
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20. The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.
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Mavromatis K, Doyle CK, Lykidis A, Ivanova N, Francino MP, Chain P, Shin M, Malfatti S, Larimer F, Copeland A, Detter JC, Land M, Richardson PM, Yu XJ, Walker DH, McBride JW, and Kyrpides NC
- Subjects
- Animals, Bacterial Proteins genetics, Dog Diseases microbiology, Dogs, Ehrlichia canis classification, Ehrlichia canis pathogenicity, Ehrlichiosis veterinary, Gene Expression Regulation, Bacterial, Glycoproteins genetics, Molecular Sequence Data, Pseudogenes, RNA, Bacterial genetics, Transcription, Genetic, Ehrlichia canis genetics, Ehrlichia canis immunology, Genome, Bacterial
- Abstract
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).
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- 2006
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21. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000.
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Feil H, Feil WS, Chain P, Larimer F, DiBartolo G, Copeland A, Lykidis A, Trong S, Nolan M, Goltsman E, Thiel J, Malfatti S, Loper JE, Lapidus A, Detter JC, Land M, Richardson PM, Kyrpides NC, Ivanova N, and Lindow SE
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins physiology, Base Composition, DNA Transposable Elements, DNA, Bacterial chemistry, DNA, Bacterial genetics, Molecular Sequence Data, Plasmids genetics, Prophages genetics, Pseudomonas syringae classification, Pseudomonas syringae pathogenicity, Pseudomonas syringae physiology, Species Specificity, Virulence genetics, Genome, Bacterial, Pseudomonas syringae genetics
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
22. How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens.
- Author
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Oren A, Larimer F, Richardson P, Lapidus A, and Csonka LN
- Subjects
- Amino Acids chemistry, Bacterial Proteins chemistry, Bacterial Proteins genetics, Halomonadaceae chemistry, Ribosomal Proteins, Genome, Bacterial genetics, Halomonadaceae drug effects, Halomonadaceae genetics, Salts pharmacology
- Abstract
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.
- Published
- 2005
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23. Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis.
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Hendrickson EL, Kaul R, Zhou Y, Bovee D, Chapman P, Chung J, Conway de Macario E, Dodsworth JA, Gillett W, Graham DE, Hackett M, Haydock AK, Kang A, Land ML, Levy R, Lie TJ, Major TA, Moore BC, Porat I, Palmeiri A, Rouse G, Saenphimmachak C, Söll D, Van Dien S, Wang T, Whitman WB, Xia Q, Zhang Y, Larimer FW, Olson MV, and Leigh JA
- Subjects
- Archaeal Proteins genetics, Methanococcus metabolism, Molecular Sequence Data, Proteome, Archaeal Proteins metabolism, Genome, Archaeal, Hydrogen metabolism, Methane metabolism, Methanococcus genetics, Sequence Analysis, DNA
- Abstract
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.
- Published
- 2004
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24. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.
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Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, Georgescu AM, Vergez LM, Land ML, Motin VL, Brubaker RR, Fowler J, Hinnebusch J, Marceau M, Medigue C, Simonet M, Chenal-Francisque V, Souza B, Dacheux D, Elliott JM, Derbise A, Hauser LJ, and Garcia E
- Subjects
- Bacteriophages genetics, Chromosome Mapping, Chromosomes, Bacterial, Gene Library, Molecular Sequence Data, Yersinia pestis metabolism, Yersinia pseudotuberculosis metabolism, Evolution, Molecular, Genome, Bacterial, Yersinia pestis genetics, Yersinia pseudotuberculosis genetics
- Abstract
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.
- Published
- 2004
- Full Text
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25. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78.
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Martinez D, Larrondo LF, Putnam N, Gelpke MD, Huang K, Chapman J, Helfenbein KG, Ramaiya P, Detter JC, Larimer F, Coutinho PM, Henrissat B, Berka R, Cullen D, and Rokhsar D
- Subjects
- Base Composition genetics, Biodegradation, Environmental, Classification, Cytochrome P-450 Enzyme System genetics, DNA, Fungal chemistry, DNA, Fungal isolation & purification, Exons genetics, Fungal Proteins classification, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Library, Genes, Fungal genetics, Genomics, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Glycosyltransferases genetics, Glycosyltransferases metabolism, Introns genetics, Laccase genetics, Laccase metabolism, Oxidoreductases genetics, Oxidoreductases metabolism, Peroxidases genetics, Peroxidases metabolism, Phanerochaete metabolism, Polysaccharides metabolism, Retroelements genetics, Sequence Analysis, DNA, Transposases genetics, Cellulose metabolism, DNA, Fungal genetics, Genome, Fungal, Lignin metabolism, Phanerochaete genetics
- Abstract
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.
- Published
- 2004
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- View/download PDF
26. Analysis and annotation of microbial genome sequences.
- Author
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Hauser L, Larimer F, Land M, Shah M, and Uberbacher E
- Subjects
- Codon, Pseudogenes, Genome, Microbiology
- Published
- 2004
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27. The genome of a motile marine Synechococcus.
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Palenik B, Brahamsha B, Larimer FW, Land M, Hauser L, Chain P, Lamerdin J, Regala W, Allen EE, McCarren J, Paulsen I, Dufresne A, Partensky F, Webb EA, and Waterbury J
- Subjects
- Bacterial Proteins genetics, Base Composition, Chromosomes, Bacterial genetics, Cyanobacteria classification, Cyanobacteria virology, Genes, Bacterial genetics, Molecular Sequence Data, Sequence Analysis, DNA, Cyanobacteria genetics, Genome, Bacterial
- Abstract
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.
- Published
- 2003
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28. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic Archaea Methanogenium frigidum and Methanococcoides burtonii.
- Author
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Saunders NF, Thomas T, Curmi PM, Mattick JS, Kuczek E, Slade R, Davis J, Franzmann PD, Boone D, Rusterholtz K, Feldman R, Gates C, Bench S, Sowers K, Kadner K, Aerts A, Dehal P, Detter C, Glavina T, Lucas S, Richardson P, Larimer F, Hauser L, Land M, and Cavicchioli R
- Subjects
- Amino Acid Sequence, Archaeal Proteins genetics, Bacterial Proteins genetics, Base Composition, DNA-Binding Proteins genetics, Genes, Archaeal genetics, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Transfer chemistry, RNA, Transfer genetics, RNA-Binding Proteins genetics, Sequence Homology, Amino Acid, Adaptation, Physiological genetics, Cold Temperature, Genome, Archaeal, Methanomicrobiaceae genetics, Methanosarcinaceae genetics
- Abstract
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.
- Published
- 2003
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29. A computational study of Shewanella oneidensis MR-1: structural prediction and functional inference of hypothetical proteins.
- Author
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Yost C, Hauser L, Larimer F, Thompson D, Beliaev A, Zhou J, Xu Y, and Xu D
- Subjects
- ATP-Binding Cassette Transporters genetics, Algorithms, Amidohydrolases genetics, Amino Acid Motifs genetics, Amino Acid Sequence, Bacterial Proteins chemistry, Carrier Proteins genetics, Chromosome Mapping methods, Conserved Sequence genetics, Databases, Factual, Databases, Nucleic Acid, Databases, Protein, Dynamins genetics, Genome, Bacterial, Heat-Shock Proteins genetics, Hydrophobic and Hydrophilic Interactions, Iron metabolism, Models, Molecular, Molecular Sequence Data, Molybdenum metabolism, Protein Conformation, Protein Folding, Protein Sorting Signals genetics, Protein Structure, Secondary, Protein Structure, Tertiary, Repressor Proteins genetics, Sequence Alignment methods, Shewanella physiology, Structural Homology, Protein, Bacterial Proteins genetics, Computational Biology, Shewanella genetics
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
30. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea.
- Author
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Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M, Hauser L, Hooper A, Klotz M, Norton J, Sayavedra-Soto L, Arciero D, Hommes N, Whittaker M, and Arp D
- Subjects
- ATP-Binding Cassette Transporters genetics, Biological Transport, Carbon Dioxide metabolism, Citric Acid metabolism, Genomics, Iron metabolism, Molecular Sequence Data, Nitrosomonas growth & development, Nitrosomonas metabolism, Oxidation-Reduction, Ammonia metabolism, Genome, Bacterial, Nitrosomonas genetics
- Abstract
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.
- Published
- 2003
- Full Text
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31. The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L.
- Author
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Gunter LE, Roberts GT, Lee K, Larimer FW, and Tuskan GA
- Subjects
- Genetic Markers, Polymerase Chain Reaction, Salix genetics, Sex Determination Processes
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
32. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.
- Author
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Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang HG, Awazu S, Azumi K, Boore J, Branno M, Chin-Bow S, DeSantis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee BI, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, and Rokhsar DS
- Subjects
- Alleles, Animals, Apoptosis, Base Sequence, Cellulose metabolism, Central Nervous System physiology, Ciona intestinalis anatomy & histology, Ciona intestinalis classification, Ciona intestinalis physiology, Computational Biology, Endocrine System physiology, Gene Dosage, Gene Duplication, Genes, Genes, Homeobox, Heart embryology, Heart physiology, Immunity genetics, Molecular Sequence Data, Multigene Family, Muscle Proteins genetics, Organizers, Embryonic physiology, Phylogeny, Polymorphism, Genetic, Proteins genetics, Proteins physiology, Sequence Homology, Nucleic Acid, Species Specificity, Thyroid Gland physiology, Urochordata genetics, Vertebrates anatomy & histology, Vertebrates classification, Vertebrates genetics, Vertebrates physiology, Ciona intestinalis genetics, Genome, Sequence Analysis, DNA
- Abstract
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.
- Published
- 2002
- Full Text
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33. Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis.
- Author
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VerBerkmoes NC, Bundy JL, Hauser L, Asano KG, Razumovskaya J, Larimer F, Hettich RL, and Stephenson JL Jr
- Subjects
- Amino Acid Sequence, Cell Fractionation, Chromatography methods, Databases, Protein, Molecular Sequence Data, Sequence Alignment, Bacterial Proteins analysis, Mass Spectrometry methods, Proteome analysis, Shewanella chemistry
- Abstract
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.
- Published
- 2002
- Full Text
- View/download PDF
34. The photosynthetic apparatus of Prochlorococcus: Insights through comparative genomics.
- Author
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Hess WR, Rocap G, Ting CS, Larimer F, Stilwagen S, Lamerdin J, and Chisholm SW
- Abstract
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.
- Published
- 2001
- Full Text
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35. The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.
- Author
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Mackenzie C, Choudhary M, Larimer FW, Predki PF, Stilwagen S, Armitage JP, Barber RD, Donohue TJ, Hosler JP, Newman JE, Shapleigh JP, Sockett RE, Zeilstra-Ryalls J, and Kaplan S
- Abstract
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.
- Published
- 2001
- Full Text
- View/download PDF
36. An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.
- Author
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Meeks JC, Elhai J, Thiel T, Potts M, Larimer F, Lamerdin J, Predki P, and Atlas R
- Abstract
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.
- Published
- 2001
- Full Text
- View/download PDF
37. HOBACGEN: homologous bacterial genes database.
- Author
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Larimer F
- Subjects
- Genomics statistics & numerical data, Internet, Sequence Alignment statistics & numerical data, Computational Biology, Databases, Factual, Genes, Bacterial, Software
- Published
- 2000
- Full Text
- View/download PDF
38. D-ribose-5-phosphate isomerase from spinach: heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme.
- Author
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Jung CH, Hartman FC, Lu TY, and Larimer FW
- Subjects
- Aldose-Ketose Isomerases chemistry, Amino Acid Sequence, Cloning, Molecular, Dimerization, Enzyme Inhibitors pharmacology, Escherichia coli, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphates pharmacology, Recombinant Proteins chemistry, Recombinant Proteins genetics, Sequence Alignment, Aldose-Ketose Isomerases genetics, Spinacia oleracea enzymology
- Abstract
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.)
- Published
- 2000
- Full Text
- View/download PDF
39. Protein threading by PROSPECT: a prediction experiment in CASP3.
- Author
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Xu Y, Xu D, Crawford OH, Einstein, Larimer F, Uberbacher E, Unseren MA, and Zhang G
- Subjects
- Amino Acid Sequence, Computer Simulation, Models, Molecular, Molecular Sequence Data, Protein Structure, Secondary, Protein Folding, Proteins chemistry, Software
- Abstract
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 (=15 A between their beta-carbons) and (iii) an alignment gap penalty; and (b) that it guarantees to find the globally-optimal alignment under various constraints on the unknown protein specified by the user. In the CASP3 experiment, PROSPECT correctly identified the most similar folds for 11 targets and predicted closely-similar folds for five other targets among the 23 targets which can be classified into the category of fold-recognition problems and also had their experimentally-determined structures available. Among the 11 correctly identified folds, PROSPECT obtained good sequence-structure alignments for nine of them. On three of the five ab initio prediction problems, PROSPECT successfully located partial structures from our template library, which align accurately with the corresponding targets.
- Published
- 1999
- Full Text
- View/download PDF
40. Identification of a catalytic aspartyl residue of D-ribulose 5-phosphate 3-epimerase by site-directed mutagenesis.
- Author
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Chen YR, Larimer FW, Serpersu EH, and Hartman FC
- Subjects
- Amino Acid Sequence, Base Sequence, Carbohydrate Epimerases chemistry, Carbohydrate Epimerases genetics, Catalytic Domain, DNA Primers, Mutagenesis, Site-Directed, Protein Structure, Secondary, Aspartic Acid metabolism, Carbohydrate Epimerases metabolism
- Abstract
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.
- Published
- 1999
- Full Text
- View/download PDF
41. Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs.
- Author
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Doktycz MJ, Larimer FW, Pastrnak M, and Stevens A
- Subjects
- Base Sequence, Fungal Proteins genetics, Molecular Sequence Data, RNA, Fungal chemical synthesis, RNA, Messenger chemical synthesis, Saccharomyces cerevisiae, Nucleic Acid Conformation, RNA, Fungal chemistry, RNA, Messenger chemistry
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
42. D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme.
- Author
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Chen YR, Hartman FC, Lu TY, and Larimer FW
- Subjects
- Base Sequence, Carbohydrate Epimerases metabolism, Cloning, Molecular, DNA, Complementary genetics, DNA, Plant genetics, Enzyme Stability, Gene Expression, Kinetics, Molecular Sequence Data, Molecular Weight, Protein Conformation, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Thermodynamics, Carbohydrate Epimerases genetics, Carbohydrate Epimerases isolation & purification, Genes, Plant, Spinacia oleracea enzymology, Spinacia oleracea genetics
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
43. Roles and microenvironments of tryptophanyl residues of spinach phosphoribulokinase.
- Author
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Brandes HK, Larimer FW, Lu TY, Dey J, and Hartman FC
- Subjects
- Amino Acid Sequence, Base Sequence, Binding Sites genetics, DNA Primers genetics, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphotransferases (Alcohol Group Acceptor) chemistry, Phosphotransferases (Alcohol Group Acceptor) genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Spectrometry, Fluorescence, Spinacia oleracea genetics, Tryptophan chemistry, Phosphotransferases (Alcohol Group Acceptor) metabolism, Spinacia oleracea enzymology
- Abstract
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.)
- Published
- 1998
- Full Text
- View/download PDF
44. Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.
- Author
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Harpel MR, Larimer FW, and Hartman FC
- Subjects
- Catalysis, Histidine chemistry, Hydrogen Bonding, Kinetics, Metalloproteins chemistry, Mutagenesis, Site-Directed, Structure-Activity Relationship, Rhodospirillum rubrum enzymology, Ribulose-Bisphosphate Carboxylase chemistry
- Abstract
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.
- Published
- 1998
- Full Text
- View/download PDF
45. Mapping individual cosmid DNAs by direct AFM imaging.
- Author
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Allison DP, Kerper PS, Doktycz MJ, Thundat T, Modrich P, Larimer FW, Johnson DK, Hoyt PR, Mucenski ML, and Warmack RJ
- Subjects
- Animals, Bacteriophage lambda genetics, Binding Sites genetics, Cloning, Molecular, DNA metabolism, Image Processing, Computer-Assisted methods, Mice, Protein Binding, Chromosome Mapping methods, Cosmids genetics, DNA genetics, Microscopy, Atomic Force methods
- Abstract
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.
- Published
- 1997
- Full Text
- View/download PDF
46. Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.
- Author
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Geck MK, Larimer FW, and Hartman FC
- Subjects
- Amino Acid Sequence, Chloroplast Thioredoxins, Enzyme Activation, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Plant Proteins chemistry, Sequence Homology, Amino Acid, Spinacia oleracea enzymology, Thioredoxins chemistry, Thioredoxins genetics, Fructose-Bisphosphatase metabolism, Malate Dehydrogenase metabolism, Plant Proteins metabolism, Spinacia oleracea metabolism, Thioredoxins metabolism
- Abstract
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.
- Published
- 1996
- Full Text
- View/download PDF
47. Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.
- Author
-
Allison DP, Kerper PS, Doktycz MJ, Spain JA, Modrich P, Larimer FW, Thundat T, and Warmack RJ
- Subjects
- Binding Sites, Deoxyribonuclease EcoRI genetics, Mutation, Chromosome Mapping methods, Deoxyribonuclease EcoRI ultrastructure, Microscopy, Atomic Force methods, Plasmids ultrastructure, Sequence Analysis methods
- Abstract
Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.
- Published
- 1996
- Full Text
- View/download PDF
48. Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis.
- Author
-
Brandes HK, Hartman FC, Lu TY, and Larimer FW
- Subjects
- Amino Acid Sequence, Base Sequence, Binding Sites genetics, Cysteine genetics, DNA Primers genetics, Gene Expression, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins genetics, Genes, Plant, Phosphotransferases (Alcohol Group Acceptor) genetics, Pichia genetics, Spinacia oleracea enzymology, Spinacia oleracea genetics
- Abstract
Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.
- Published
- 1996
- Full Text
- View/download PDF
49. The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f.
- Author
-
Brandes HK, Larimer FW, and Hartman FC
- Subjects
- Binding Sites, Chloroplast Thioredoxins, Cysteine, Disulfides, Electrophoresis, Polyacrylamide Gel, Macromolecular Substances, Mutagenesis, Site-Directed, Phosphotransferases (Alcohol Group Acceptor) chemistry, Phosphotransferases (Alcohol Group Acceptor) isolation & purification, Point Mutation, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sulfhydryl Reagents pharmacology, Thioredoxins chemistry, Thioredoxins isolation & purification, Phosphotransferases (Alcohol Group Acceptor) metabolism, Plants enzymology, Thioredoxins metabolism
- Abstract
Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schürmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx.
- Published
- 1996
- Full Text
- View/download PDF
50. Analysis of spontaneous frameshift mutations in REV1 and rev1-1 strains of Saccharomyces cerevisiae.
- Author
-
Kalinowski DP, Larimer FW, and Plewa MJ
- Subjects
- Base Sequence, DNA, Fungal chemistry, Molecular Sequence Data, Polymerase Chain Reaction, Frameshift Mutation, Saccharomyces cerevisiae genetics
- Abstract
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.
- Published
- 1995
- Full Text
- View/download PDF
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