David A. Mead

Assembly of viral metagenomes from yellowstone hot springs.

ABSTRACT Thermophilic viruses were reported decades ago; however, knowledge of their diversity, biology, and ecological impact is limited. Previous research on thermophilic viruses focused on cultivated strains. This study examined metagenomic profiles of viruses directly isolated from two mildly alkaline hot springs, Bear Paw (74°C) and Octopus (93°C). Using a new method for constructing libraries from picograms of DNA, nearly 30 Mb of viral DNA sequence was determined. In contrast to previous studies, sequences were assembled at 50% and 95% identity, creating composite contigs up to 35 kb and facilitating analysis of the inherent heterogeneity in the populations. Lowering the assembly identity reduced the estimated number of viral types from 1,440 and 1,310 to 548 and 283, respectively. Surprisingly, the diversity of viral species in these springs approaches that in moderate-temperature environments. While most known thermophilic viruses have a chronic, nonlytic infection lifestyle, analysis of coding sequences suggests lytic viruses are more common in geothermal environments than previously thought. The 50% assembly included one contig with high similarity and perfect synteny to nine genes from Pyrobaculum spherical virus (PSV). In fact, nearly all the genes of the 28-kb genome of PSV have apparent homologs in the metagenomes. Similarities to thermoacidophilic viruses isolated on other continents were limited to specific open reading frames but were equally strong. Nearly 25% of the reads showed significant similarity between the hot springs, suggesting a common subterranean source. To our knowledge, this is the first application of metagenomics to viruses of geothermal origin.

The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist.

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

Functional viral metagenomics and the next generation of molecular tools

The enzymes of bacteriophages and other viruses have been essential research tools since the first days of molecular biology. However, the current repertoire of viral enzymes only hints at their overall potential. The most commonly used enzymes are derived from a surprisingly small number of cultivated viruses, which is remarkable considering the extreme abundance and diversity of viruses revealed over the past decade by metagenomic analysis. To access the treasure trove of enzymes hidden in the global virosphere and develop them for research, therapeutic and diagnostic uses, improvements are needed in our ability to rapidly and efficiently discover, express and characterize viral genes to produce useful proteins. In this paper, we discuss improvements to sampling and cloning methods, functional and genomics-based screens, and expression systems, which should accelerate discovery of new enzymes and other viral proteins for use in research and medicine.

Complete Genome of the Cellulolytic Ruminal Bacterium Ruminococcus albus 7

Ruminococcus albus 7 is a highly cellulolytic ruminal bacterium that is a member of the phylum Firmicutes. Here, we describe the complete genome of this microbe. This genome will be useful for rumen microbiology and cellulosome biology and in biofuel production, as one of its major fermentation products is ethanol.

A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP)

Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes, Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas, and Proposal of Cellulomonas gilvus sp. nov

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.

Molecular Cloning of the Three Base Restriction Endonuclease R.CviJI from Eukaryotic Chlorella Virus IL-3A

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.

Mining Dictyoglomus turgidum for Enzymatically Active Carbohydrases

The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidum does not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.

Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases:

BackgroundTo efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms.ResultsFrom the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids.ConclusionsMetagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.