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1.  The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis 
BMC Microbiology  2014;14:94.
Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type.
Relative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings.
PtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.
PMCID: PMC3997438  PMID: 24739259
Antifungal; Biocontrol; Chitinase; Motility; Phenazine; Pseudomonas; Siderophore; Transcriptional regulator
2.  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
3.  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

Results 1-3 (3)