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Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis.

ABSTRACT: The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.

SUBMITTER: Lochner A

PROVIDER: S-EPMC3131623 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis.

Lochner Adriane A Giannone Richard J RJ Rodriguez Miguel M Shah Manesh B MB Mielenz Jonathan R JR Keller Martin M Antranikian Garabed G Graham David E DE Hettich Robert L RL

Applied and environmental microbiology 20110415 12

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative g ...[more]

PMID: 21498747

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Project description:BackgroundLabel-free quantitative proteomics holds a great deal of promise for the future study of both medicine and biology. However, the data generated is extremely intricate in its correlation structure, and its proper analysis is complex. There are issues with missing identifications. There are high levels of correlation between many, but not all, of the peptides derived from the same protein. Additionally, there may be systematic shifts in the sensitivity of the machine between experiments or even through time within the duration of a single experiment.ResultsWe describe a hierarchical model for analyzing unbiased, label-free proteomics data which utilizes the covariance of peptide expression across samples as well as MS/MS-based identifications to group peptides-a strategy we call metaprotein expression modeling. Our metaprotein model acknowledges the possibility of misidentifications, post-translational modifications and systematic differences between samples due to changes in instrument sensitivity or differences in total protein concentration. In addition, our approach allows us to validate findings from unbiased, label-free proteomics experiments with further unbiased, label-free proteomics experiments. Finally, we demonstrate the clinical/translational utility of the model for building predictors capable of differentiating biological phenotypes as well as for validating those findings in the context of three novel cohorts of patients with Hepatitis C.ConclusionsMass-spectrometry proteomics is quickly becoming a powerful tool for studying biological and translational questions. Making use of all of the information contained in a particular set of data will be critical to the success of those endeavors. Our proposed model represents an advance in the ability of statistical models of proteomic data to identify and utilize correlation between features. This allows validation of predictors without translation to targeted assays in addition to informing the choice of targets when it is appropriate to generate those assays.

Project description:BackgroundBiogas production from lignocellulosic biomass is generally considered to be challenging due to the recalcitrant nature of this biomass. In this study, the recalcitrance of birch was reduced by applying steam-explosion (SE) pretreatment (210 °C and 10 min). Moreover, bioaugmentation with the cellulolytic bacterium Caldicellulosiruptor bescii was applied to possibly enhance the methane production from steam-exploded birch in an anaerobic digestion (AD) process under thermophilic conditions (62 °C).ResultsOverall, the combined SE and bioaugmentation enhanced the methane yield up to 140% compared to untreated birch, while SE alone contributed to the major share of methane enhancement by 118%. The best methane improvement of 140% on day 50 was observed in bottles fed with pretreated birch and bioaugmentation with lower dosages of C. bescii (2 and 5% of inoculum volume). The maximum methane production rate also increased from 4-mL CH4/g VS (volatile solids)/day for untreated birch to 9-14-mL CH4/g VS/day for steam-exploded birch with applied bioaugmentation. Bioaugmentation was particularly effective for increasing the initial methane production rate of the pretreated birch yielding 21-44% more methane than the pretreated birch without applied bioaugmentation. The extent of solubilization of the organic matter was increased by more than twofold when combined SE pretreatment and bioaugmentation was used in comparison with the methane production from untreated birch. The beneficial effects of SE and bioaugmentation on methane yield indicated that biomass recalcitrance and hydrolysis step are the limiting factors for efficient AD of lignocellulosic biomass. Microbial community analysis by 16S rRNA amplicon sequencing showed that the microbial community composition was altered by the pretreatment and bioaugmentation processes. Notably, the enhanced methane production by pretreatment and bioaugmentation was well correlated with the increase in abundance of key bacterial and archaeal communities, particularly the hydrolytic bacterium Caldicoprobacter, several members of syntrophic acetate oxidizing bacteria and the hydrogenotrophic Methanothermobacter.ConclusionOur findings demonstrate the potential of combined SE and bioaugmentation for enhancing methane production from lignocellulosic biomass.

Project description:Background:Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results:Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions-introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions:Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

Dataset Information

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis.

Publications

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis.

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