Engineering and characterization of new LOV-based fluorescent proteins from Chlamydomonas reinhardtii and Vaucheria frigida.

Flavin-based fluorescent proteins (FbFPs) are a new class of fluorescent reporters that exhibit oxygen-independent fluorescence, which is a key advantage over the green fluorescent protein. Broad application of FbFPs, however, has been generally hindered by low brightness. To maximize the utility of FbFPs, there is a pressing need to expand and diversify the limited FbFP library through the inclusion of bright and robust variants.

Nitrogen utilization and metabolism in Ruminococcus albus 8.

The model rumen Firmicutes organism Ruminococcus albus 8 was grown using ammonia, urea, or peptides as the sole nitrogen source; growth was not observed with amino acids as the sole nitrogen source. Growth of R. albus 8 on ammonia and urea showed the same growth rate (0.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure).

Probiotic dosing of Ruminococcus flavefaciens affects rumen microbiome structure and function in reindeer.

Highly cellulolytic bacterial species such as Ruminococcus flavefaciens are regarded essential for the microbial breakdown of cellulose in the rumen. We have investigated the effect of ruminal dosing of R. flavefaciens strain 8/94-32 during realimentation of starved reindeer (males, n = 3).

Insights into lignin degradation and its potential industrial applications.

Lignocellulose is an abundant biomass that provides an alternative source for the production of renewable fuels and chemicals. The depolymerization of the carbohydrate polymers in lignocellulosic biomass is hindered by lignin, which is recalcitrant to chemical and biological degradation due to its complex chemical structure and linkage heterogeneity. The role of fungi in delignification due to the production of extracellular oxidative enzymes has been studied more extensively than that of bacteria.

Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars.

Heterologous gene expression in filamentous fungi.

Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms.

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C.

Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaerobius polysaccharolyticus.

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus.

Molecular and biochemical analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii.

A large polypeptide encoded in the genome of the thermophilic bacterium Caldicellulosiruptor bescii was determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X.

Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging.

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis.

Biochemical and mutational analyses of a multidomain cellulase/mannanase from Caldicellulosiruptor bescii.

Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C.

Methanosarcina acetivorans C2A topoisomerase IIIα, an archaeal enzyme with promiscuity in divalent cation dependence.

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases.

Purification, characterization, and expression of multiple glutamine synthetases from Prevotella ruminicola 23.

The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa.

Functional analyses of multiple lichenin-degrading enzymes from the rumen bacterium Ruminococcus albus 8.

Ruminococcus albus 8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence of R. albus revealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds.

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen.

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer.

Biochemical characterization and relative expression levels of multiple carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate.

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS).

Biochemical analyses of multiple endoxylanases from the rumen bacterium Ruminococcus albus 8 and their synergistic activities with accessory hemicellulose-degrading enzymes.

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R.

Genomic fingerprinting and serotyping of Salmonella from Galápagos iguanas demonstrates island differences in strain diversity.

Salmonella carriage patterns in wild and captive reptiles suggest that both geographical proximity and host ecological differences may determine bacterial diversity among reptile populations. In this study, we explore the relative importance of these factors on Salmonella diversity in free-living Galápagos iguanas. We isolated Salmonella enterica from marine iguanas (Amblyrhynchus cristatus) and land iguanas (Conolophus subcristatus and C.

Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing.

Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species--the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation.

Structural and functional analyses of a glycoside hydrolase family 5 enzyme with an unexpected β-fucosidase activity.

We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B(1)4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a β-D-fucosidase.

Reverse-chaperoning activity of an AAA+ protein.

Speed and processivity of replicative DNA polymerases can be enhanced via coupling to a sliding clamp. Due to the closed ring shape of the clamp, a clamp loader protein, belonging to the AAA+ class of ATPases, needs to open the ring-shaped clamp before loading it to DNA. Here, we developed real-time fluorescence assays to study the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans.

Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes.

Microbial inhabitants of the bovine rumen fulfil the majority of the normal caloric requirements of the animal by fermenting lignocellulosic plant polysaccharides and releasing short chain fatty acids that are then metabolized by the host. This process also occurs within the human colon, although the fermentation products contribute less to the overall energy requirements of the host. Mounting evidence, however, indicates that the community structure of the distal gut microbiota is a critical factor that influences the inflammatory potential of the immune system thereby impacting the progression of inflammatory bowel diseases.

Response of archaeal communities in the rhizosphere of maize and soybean to elevated atmospheric CO2 concentrations.

Background: Archaea are important to the carbon and nitrogen cycles, but it remains uncertain how rising atmospheric carbon dioxide concentrations ([CO(2)]) will influence the structure and function of soil archaeal communities.

Methodology/principal Findings: We measured abundances of archaeal and bacterial 16S rRNA and amoA genes, phylogenies of archaeal 16S rRNA and amoA genes, concentrations of KCl-extractable soil ammonium and nitrite, and potential ammonia oxidation rates in rhizosphere soil samples from maize and soybean exposed to ambient (∼385 ppm) and elevated (550 ppm) [CO(2)] in a replicated and field-based study. There was no influence of elevated [CO(2)] on copy numbers of archaeal or bacterial 16S rRNA or amoA genes, archaeal community composition, KCl-extractable soil ammonium or nitrite, or potential ammonia oxidation rates for samples from maize, a model C(4) plant.

Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice.

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP).