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1.  Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates 
Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.
PMCID: PMC3957603  PMID: 24362431
2.  Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1,3-propanediol synthesis at high glycerol loading 
AMB Express  2014;4:63.
Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.
PMCID: PMC4230902  PMID: 25401066
Clostridium butyricum; 1,3-propanediol synthesis; Glycerol catabolism; Proteomics; Multiple reaction monitoring
3.  Comparative Analysis of Carbohydrate Active Enzymes in Clostridium termitidis CT1112 Reveals Complex Carbohydrate Degradation Ability 
PLoS ONE  2014;9(8):e104260.
Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.
PMCID: PMC4125193  PMID: 25101643
4.  Enhanced whole genome sequence and annotation of Clostridium stercorarium DSM8532T using RNA-seq transcriptomics and high-throughput proteomics 
BMC Genomics  2014;15(1):567.
Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532T is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics.
A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose.
Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-567) contains supplementary material, which is available to authorized users.
PMCID: PMC4102724  PMID: 24998381
Genome; Proteome; Transcriptome; RNA-seq; Tandem mass spectrometry; Proteogenomics; Glycolysis; Pentose phosphate pathway; Transaldolase
5.  Genome features of Pseudomonas putida LS46, a novel polyhydroxyalkanoate producer and its comparison with other P. putida strains 
AMB Express  2014;4:37.
A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation. Genes for toluene or naphthalene degradation found in the genomes of P. putida F1, DOT-T1E, and ND6 were absent in the P. putida LS46 genome. Heavy metal resistant genes encoded by the P. putida W619 genome were also not present in the P. putida LS46 genome. Despite the overall similarity among genome of P.putida strains isolated for different applications and from different geographical location a number of differences were observed in genome arrangement, occurrence of transposon, genomic islands and prophage. It appears that P.putida strains had a common ancestor and by acquiring some specific genes by horizontal gene transfer it differed from other related strains.
PMCID: PMC4230813  PMID: 25401060
Pseudomonas putida; Comparative bioinformatics analysis; Comparative genome analysis; Polyhydroxyalkanoates; Pan-Genome; Insertion sequences; Metabolic diversity
6.  Draft Genome Sequence of the Hydrogen- and Ethanol-Producing Bacterium Clostridium intestinale Strain URNW 
Genome Announcements  2013;1(5):e00871-13.
Here, we report the draft genome sequence of Clostridium intestinale strain URNW, which can convert biomass to useful products such as biofuels (hydrogen or ethanol) and other soluble end products.
PMCID: PMC3798459  PMID: 24136853
7.  Draft Genome Sequence of the Cellulolytic, Mesophilic, Anaerobic Bacterium Clostridium termitidis Strain CT1112 (DSM 5398) 
Genome Announcements  2013;1(3):e00281-13.
Here, we report the draft genome sequence of Clostridium termitidis strain CT1112 (DSM 5398), a mesophilic, cellulolytic bacterium that can utilize a variety of sugars, as well as pure cellulose, as a sole carbon source; it also synthesizes fermentation end products with potential industrial applications.
PMCID: PMC3662827  PMID: 23704187
8.  Draft Genome Sequence of Medium-Chain-Length Polyhydroxyalkanoate-Producing Pseudomonas putida Strain LS46 
Genome Announcements  2013;1(2):e00151-13.
We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that synthesizes medium-chain-length polyhydroxyalkanoates. The draft genome of P. putida LS46 consists of approximately 5.86 million bp, with a G+C content of 61.69%. A total of 5,316 annotated genes and 5,219 coding sequences (CDS) were identified.
PMCID: PMC3630404  PMID: 23599293
9.  Genomic Evaluation of Thermoanaerobacter spp. for the Construction of Designer Co-Cultures to Improve Lignocellulosic Biofuel Production 
PLoS ONE  2013;8(3):e59362.
The microbial production of ethanol from lignocellulosic biomass is a multi-component process that involves biomass hydrolysis, carbohydrate transport and utilization, and finally, the production of ethanol. Strains of the genus Thermoanaerobacter have been studied for decades due to their innate abilities to produce comparatively high ethanol yields from hemicellulose constituent sugars. However, their inability to hydrolyze cellulose, limits their usefulness in lignocellulosic biofuel production. As such, co-culturing Thermoanaerobacter spp. with cellulolytic organisms is a plausible approach to improving lignocellulose conversion efficiencies and yields of biofuels. To evaluate native lignocellulosic ethanol production capacities relative to competing fermentative end-products, comparative genomic analysis of 11 sequenced Thermoanaerobacter strains, including a de novo genome, Thermoanaerobacter thermohydrosulfuricus WC1, was conducted. Analysis was specifically focused on the genomic potential for each strain to address all aspects of ethanol production mentioned through a consolidated bioprocessing approach. Whole genome functional annotation analysis identified three distinct clades within the genus. The genomes of Clade 1 strains encode the fewest extracellular carbohydrate active enzymes and also show the least diversity in terms of lignocellulose relevant carbohydrate utilization pathways. However, these same strains reportedly are capable of directing a higher proportion of their total carbon flux towards ethanol, rather than non-biofuel end-products, than other Thermoanaerobacter strains. Strains in Clade 2 show the greatest diversity in terms of lignocellulose hydrolysis and utilization, but proportionately produce more non-ethanol end-products than Clade 1 strains. Strains in Clade 3, in which T. thermohydrosulfuricus WC1 is included, show mid-range potential for lignocellulose hydrolysis and utilization, but also exhibit extensive divergence from both Clade 1 and Clade 2 strains in terms of cellular energetics. The potential implications regarding strain selection and suitability for industrial ethanol production through a consolidated bioprocessing co-culturing approach are examined throughout the manuscript.
PMCID: PMC3608648  PMID: 23555660
10.  Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria 
BMC Microbiology  2012;12:295.
Fermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae.
Bioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do.
Here we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.
PMCID: PMC3561251  PMID: 23249097
11.  Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression 
BMC Microbiology  2012;12:214.
Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.
Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.
Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.
PMCID: PMC3492117  PMID: 22994686
12.  Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405 
Indian Journal of Microbiology  2008;48(2):252-266.
Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H2 and CO2. Very little is known about the factors that determine metabolic fluxes influencing H2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identified genes encoding key enzymes in pyruvate catabolism and H2-synthesis pathways, and have confirmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as “Energy-converting Hydrogenases”, or via NAD(P)Hdependent Fe-only hydrogenases which would permit H2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our findings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H2 synthesis is the first step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confirmed using both expression analysis and protein characterization.
PMCID: PMC3450175  PMID: 23100718
Clostridium thermocellum; Fermentation; Cellulose; Hydrogen; Pyruvate catabolism
13.  Effect of pH on Intracellular Accumulation of Trace Concentrations of Hg(II) in Escherichia coli under Anaerobic Conditions, as Measured Using a mer-lux Bioreporter▿  
The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.
PMCID: PMC2227699  PMID: 18083863
14.  Third Generation Biofuels via Direct Cellulose Fermentation 
Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms. CBP offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs, and higher conversion efficiencies than separate hydrolysis and fermentation processes, and is an economically attractive near-term goal for “third generation” biofuel production. In this review article, production of third generation biofuels from cellulosic feedstocks will be addressed in respect to the metabolism of cellulolytic bacteria and the development of strategies to increase biofuel yields through metabolic engineering.
PMCID: PMC2635718  PMID: 19325807
biofuels; ethanol; hydrogen; cellulose; fermentation
15.  Isolation and Ultrastructure of the Flagella of Methanococcus thermolithotrophicus and Methanospirillum hungatei 
The flagella of the archaebacteria Methanococcus thermolithotrophicus and Methanospirillum hungatei enter the cells in regions with ultrastructure resembling that of the polar organelles found in a variety of eubacteria. Flagella of both organisms consist of a filament, a hook, and a basal body with two rings similar to those of gram-positive eubacteria. The integrity of the flagella of M. thermolithotrophicus is lost in the absence of high salt concentrations, and those of both organisms are unstable at high pH. The flagellar filaments of M. hungatei are composed of two flagellins of 24 and 26 kilodaltons.
PMCID: PMC202880  PMID: 16347934

Results 1-15 (15)