Deletion Mutants, Archived Transposon Library, and Tagged Protein Constructs of the Model Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough.

The dissimilatory sulfate-reducing Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens.

Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production.

Background: The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors.

Results: We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16.

Biochemical and structural studies of NADH-dependent FabG used to increase the bacterial production of fatty acids under anaerobic conditions.

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology.

Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones.

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB).

Functionalizing bacterial cell surfaces with a phage protein.

Functionalization of bacterial cell surfaces has the potential to introduce new activities by chemical modification. Here we show that a bacteriophage-receptor complex can be used to functionalize the surface of two Gram-negative proteobacteria, Escherichia coli and Ralstonia eutropha with CdSe/ZnS nanoparticles. This work highlights the potential for using microbe-phage interactions to generate new functions on living cells.

Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in Escherichia coli.

Background: Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E.

Tracing determinants of dual substrate specificity in glycoside hydrolase family 5.

Enzymes are traditionally viewed as having exquisite substrate specificity; however, recent evidence supports the notion that many enzymes have evolved activities against a range of substrates. The diversity of activities across glycoside hydrolase family 5 (GH5) suggests that this family of enzymes may contain numerous members with activities on multiple substrates. In this study, we combined structure- and sequence-based phylogenetic analysis with biochemical characterization to survey the prevalence of dual specificity for glucan- and mannan-based substrates in the GH5 family.

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases.

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003).

Towards a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough.

Protein-protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study Escherichia coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio vulgaris Hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. Here we carried out affinity purification followed by mass spectrometry to reconstruct an interaction network among 12 chromosomally encoded bait and 90 prey proteins based on 134 bait-prey interactions identified to be of high confidence.

Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima.

Tm_Cel5A, which belongs to family 5 of the glycoside hydrolases, is an extremely stable enzyme among the endo-acting glycosidases present in the hyperthermophilic organism Thermotoga maritima. Members of GH5 family shows a common (β/α)(8) TIM-barrel fold in which the catalytic acid/base and nucleophile are located on strands β-4 and β-7 of the barrel fold. Thermally resistant cellulases are desirable for lignocellulosic biofuels production and the Tm_Cel5A is an excellent candidate for use in the degradation of polysaccharides present on biomass.

An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima.

Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixed-model analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T.

Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus.

Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima MSB8 was grown on a variety of carbohydrates to determine the influence of carbon and energy source on differential gene expression. Despite the fact that T. maritima has been phylogenetically characterized as a primitive microorganism from an evolutionary perspective, results here suggest that it has versatile and discriminating mechanisms for regulating and effecting complex carbohydrate utilization.

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM).

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C.