A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3 s per read on a Mac Mini with strain-level resolution for sequences with >84% identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95 ± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS).

Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies' (ONT) cloud-based What's In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT's framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC's 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT's MinION portability, ease-of-use, and identification capability in remote locations.

Occurrence of Bacillus amyloliquefaciens as a systemic endophyte of vanilla orchids.

We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids (Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia × V. pompona) as a systemic bacterial endophyte. We determined with light microscopy and isolations that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial endophyte and that shoot meristems and stomatal areas of stems and leaves were densely colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence data. Since additional endophyte-free plants and seed of this orchid were not available, additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea tricolor, and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens demonstrated intracellular colonization of guard cells and other epidermal cells, confirming the pattern observed in the orchids. Isolations and histological studies suggest that the bacterium may penetrate deeply into developing plant tissues in shoot meristems, forming endospores in maturing tissues. B. amyloliquefaciens produced fungal inhibitors in culture. In controlled experiments using morning glory seedlings we showed that the bacterium promoted seedling growth and reduced seedling necrosis due to pathogens. We detected the gene for phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of antifungal lipopeptides, and purified the lipopeptide "surfactin" from cultures of the bacterium. We hypothesize that B. amyloliquefaciens is a robust endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between this bacterium and its hosts can be managed to protect vanilla crops from diseases is a question that should be evaluated in future research.

Mechanisms of c-reactive protein up-regulation in arterialized vein grafts.

BACKGROUND: C-reactive protein (CRP), an acute phase reactant, is an independent predictor of coronary artery syndromes and a mediator of the vascular response to injury. CRP has been found in arterialized vein grafts and has been linked to atherogenesis; however, its involvement in vein graft early failure or intimal hyperplasia has not been assessed. This study was designed to investigate the mechanism(s) of CRP up-regulation in arterialized vein grafts. METHODS: Carotid artery bypass with arterialized jugular vein grafts (AVG) was performed in 18 dogs. AVG were harvested at 3, 8, and 24 hours and 4, 14, and 28 days, using the femoral vein obtained at the time of AVG harvest as a control. Serum CRP levels were characterized by enzyme-linked immunosorbent assay; AVG expression of CRP was studied by immunofluorescence, Western blotting, in situ hybridization, Northern blotting, and quantitative RT-PCR. RESULTS: CRP levels peaked at 24 hours in serum and AVG but remained at baseline in control veins. By double immunofluorescence, CRP was associated with the media and adventitia of AVG. However, Northern blotting analysis showed no CRP mRNA expression in AVG. Reverse transcriptase polymerase chain reaction analysis confirmed the lack of up-regulation of CRP in AVG. CONCLUSION: CRP levels are increased in AVG, peaking 24 hours after arterialization. However, no significant production of CRP was detected in AVG. Therefore, increased CRP levels within AVG appear to originate mostly from CRP diffusion from the systemic circulation. These results have significant implications for the development of strategies aimed at blocking CRP up-regulation in bypass grafts.

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7.

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.