Publications by authors named "Theodore Larson Freeman"

5 Publications

  • Page 1 of 1

Deciphering diatom biochemical pathways via whole-cell proteomics.

Aquat Microb Ecol 2009 Jun;55(3):241-253

Medicinal Chemistry Department, University of Washington, Box 335351, Seattle, Washington 98195, USA.

Diatoms play a critical role in the oceans' carbon and silicon cycles; however, a mechanistic understanding of the biochemical processes that contribute to their ecological success remains elusive. Completion of the Thalassiosira pseudonana genome provided 'blueprints' for the potential biochemical machinery of diatoms, but offers only a limited insight into their biology under various environmental conditions. Using high-throughput shotgun proteomics, we identified a total of 1928 proteins expressed by T. pseudonana cultured under optimal growth conditions, enabling us to analyze this diatom's primary metabolic and biosynthetic pathways. Of the proteins identified, 70% are involved in cellular metabolism, while 11% are involved in the transport of molecules. We identified all of the enzymes involved in the urea cycle, thereby describing the complete pathway to convert ammonia to urea, along with urea transporters, and the urea-degrading enzyme urease. Although metabolic exchange between these pathways remains ambiguous, their constitutive presence suggests complex intracellular nitrogen recycling. In addition, all C(4) related enzymes for carbon fixation have been identified to be in abundance, with high protein sequence coverage. Quantification of mass spectra acquisitions demonstrated that the 20 most abundant proteins included an unexpectedly high expression of clathrin, which is the primary structural protein involved in endocytic transport. This result highlights a previously overlooked mechanism for the inter- and intra-cellular transport of nutrients and macromolecules in diatoms, potentially providing a missing link to organelle communication and metabolite exchange. Our results demonstrate the power of proteomics, and lay the groundwork for future comparative proteomic studies and directed analyses of specifically expressed proteins and biochemical pathways of oceanic diatoms.
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http://dx.doi.org/10.3354/ame01284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2761042PMC
June 2009

Analysis of the genome of the Escherichia coli O157:H7 2006 spinach-associated outbreak isolate indicates candidate genes that may enhance virulence.

Infect Immun 2009 Sep 29;77(9):3713-21. Epub 2009 Jun 29.

Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, USA.

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.
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http://dx.doi.org/10.1128/IAI.00198-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738036PMC
September 2009

Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.

Anal Chem 2008 Nov 24;80(22):8799-806. Epub 2008 Oct 24.

Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
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http://dx.doi.org/10.1021/ac801646fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228852PMC
November 2008

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.

Genome Biol 2007 ;8(6):R102

Department of Genome Sciences, University of Washington, Campus Box 357710, 1705 NE Pacific street Seattle, Washington 98195, USA.

Background: Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.

Results: Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.

Conclusion: The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.
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http://dx.doi.org/10.1186/gb-2007-8-6-r102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394750PMC
February 2008

Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients.

Mol Microbiol 2007 Apr;64(2):512-33

Department of Microbiology, University of Washington, Seattle, WA, USA.

The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased beta-lactamase activity that increased tolerance to ceftazidime, a widely used beta-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05678.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742308PMC
April 2007
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