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Search Results: 1 - 10 of 232062 matches for " Robert L. Hettich "
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Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose
Kumaran Sivagnanam, Vijaya GS Raghavan, Manesh Shah, Robert L Hettich, Nathan C Verberkmoes, Mark G Lefsrud
Proteome Science , 2011, DOI: 10.1186/1477-5956-9-66
Abstract: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates.Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.Clostridium acetobutylicum is a gram positive, spore forming, obligate anaerobic bacteria and is one of the few microorganisms capable of converting a wide variety of sugars into three main products acetone, butanol and ethanol (ABE) [1]. ABE fermentation process was the primary source of butanol for over 40 years until the mid-1950s and is one of the oldest large-scale industrial fermentations [2]. ABE fermentation could not compete with the chemical synthesis of ABE solvents from petroleum since the mid-1950s [3]. However, increased concern over depletion of fossil fuels has led to renewed research interest in producing solvents via microbial fermentation processes.Lignocellulosic biomass is an abundant renewable resource that can be used for the production of alternative fuels [4]. It is advantageous to use lignocellulosic biomass such as rice straw, wheat straw, corn stover and agricultural residues for biofuel production as they have limited impact on food supplies [5]. Glucose is the most abundant sugar fou
Phenotype Fingerprinting Suggests the Involvement of Single-Genotype Consortia in Degradation of Aromatic Compounds by Rhodopseudomonas palustris
Tatiana V. Karpinets, Dale A. Pelletier, Chongle Pan, Edward C. Uberbacher, Galina V. Melnichenko, Robert L. Hettich, Nagiza F. Samatova
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004615
Abstract: Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N2-fixing subpopulation in the benzoate degrading consortium has preferential activation of the vanadium nitrogenase over the molybdenum nitrogenase. This subpopulation in the consortium was confirmed in an independent experiment by consumption of dissolved nitrogen gas under the benzoate degrading conditions.
Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans-Ignicoccus hospitalis Relationship
Richard J. Giannone, Harald Huber, Tatiana Karpinets, Thomas Heimerl, Ulf Küper, Reinhard Rachel, Martin Keller, Robert L. Hettich, Mircea Podar
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0022942
Abstract: Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis.
Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus
Jacque C. Young, Brian D. Dill, Chongle Pan, Robert L. Hettich, Jillian F. Banfield, Manesh Shah, Christophe Fremaux, Philippe Horvath, Rodolphe Barrangou, Nathan C. VerBerkmoes
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038077
Abstract: The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.
A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry
Chongle Pan, Byung H Park, William H McDonald, Patricia A Carey, Jillian F Banfield, Nathan C VerBerkmoes, Robert L Hettich, Nagiza F Samatova
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-118
Abstract: In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy.Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode webcite.Tandem mass spectrometry (MS/MS) has become an important method for characterizing complex protein mixtures. Since the emergence of this high-throughput technology, two complementary data analysis approaches have been pursued: a database searching approach and a de novo sequencing approach. The former identifies peptide sequences from a protein database by matching their predicted tandem mass spectra to measured tandem mass spectra; whereas the latter infers partial or complete peptide sequences directly from measured tandem mass spectra. Due to its high identification accuracy and the rapid expansion of genomic sequence data, the database searching approach, enabled by popular algorithms such as Sequest [1] and Mascot [2], is routinely used in current proteomics workflows. In contrast, although many de novo algorithms have been developed [3-14], they have not been widely used in high-throughput proteomics workflows. A high-throughput de novo seq
Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge
Dorothea K Thompson, Karuna Chourey, Gene S Wickham, Stephanie B Thieman, Nathan C VerBerkmoes, Bing Zhang, Andrea T McCarthy, Matt A Rudisill, Manesh Shah, Robert L Hettich
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-311
Abstract: Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions.Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities.Pseudomonas putida is a ubiquitous gram-negative, saprophytic bacterium belonging to the gamma class of the Proteobacteria. Endowed with a remarkable environmental adaptability, P. putida strain F1 [1], for example, has been investigated most extensively as a model organism for the microbial degradation of such xenobiotic aromatic compounds as toluene, benzene, and ethylbenzene [2]. Considerably less scientific focus
Integrated Metagenomics/Metaproteomics Reveals Human Host-Microbiota Signatures of Crohn's Disease
Alison R. Erickson, Brandi L. Cantarel, Regina Lamendella, Youssef Darzi, Emmanuel F. Mongodin, Chongle Pan, Manesh Shah, Jonas Halfvarson, Curt Tysk, Bernard Henrissat, Jeroen Raes, Nathan C. Verberkmoes, Claire M. Fraser, Robert L. Hettich, Janet K. Jansson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049138
Abstract: Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.
Strategies for Metagenomic-Guided Whole-Community Proteomics of Complex Microbial Environments
Brandi L. Cantarel, Alison R. Erickson, Nathan C. VerBerkmoes, Brian K. Erickson, Patricia A. Carey, Chongle Pan, Manesh Shah, Emmanuel F. Mongodin, Janet K. Jansson, Claire M. Fraser-Liggett, Robert L. Hettich
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0027173
Abstract: Accurate protein identification in large-scale proteomics experiments relies upon a detailed, accurate protein catalogue, which is derived from predictions of open reading frames based on genome sequence data. Integration of mass spectrometry-based proteomics data with computational proteome predictions from environmental metagenomic sequences has been challenging because of the variable overlap between proteomic datasets and corresponding short-read nucleotide sequence data. In this study, we have benchmarked several strategies for increasing microbial peptide spectral matching in metaproteomic datasets using protein predictions generated from matched metagenomic sequences from the same human fecal samples. Additionally, we investigated the impact of mass spectrometry-based filters (high mass accuracy, delta correlation), and de novo peptide sequencing on the number and robustness of peptide-spectrum assignments in these complex datasets. In summary, we find that high mass accuracy peptide measurements searched against non-assembled reads from DNA sequencing of the same samples significantly increased identifiable proteins without sacrificing accuracy.
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome
Nathan P. McNulty,Meng Wu,Alison R. Erickson,Chongle Pan,Brian K. Erickson,Eric C. Martens,Nicholas A. Pudlo,Brian D. Muegge,Bernard Henrissat,Robert L. Hettich,Jeffrey I. Gordon
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001637
Abstract: The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.
Heinrich’s Fourth Dimension  [PDF]
Robert L. Collins
Open Journal of Safety Science and Technology (OJSST) , 2011, DOI: 10.4236/ojsst.2011.11003
Abstract: In this article, the author uses accident data readily available from the United States Bureau of Labor Statistics to create a New Incident Pyramid, a modern day equivalent of Heinrich’s Triangle. This historical data is then combined with generally accepted statistical methods to show that the relationship between incident types first envisioned by Herbert William Heinrich in 1931 has a fourth dimension, time. Using statistical analysis methods derived from both a Binomial distribution and a Poisson distribution, this analysis will show how information derived from these accident summaries can be used to predict potential future events. The obvious conclusion reinforced by this analysis will be that the future date for potentially fatal accidents can only be delayed by focusing on accident prevention strategies that address all incidents without regard for the type of resulting injury. This analysis can be used by safety professionals to predict potential future outcomes for their establishments which can then be used to better communicate the need for improvements in accident prevention programs.
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