Sequence of primers used in this study.

<p>Sequence of primers used in this study.</p

Recombinant MT0516 exhibits concentration- and time-dependent exopolyphosphatase activity.

<p>A. Polyacrylamide gel electrophoresis showing the fractions of recombinant His-tagged MT0516 protein (36.6 KDa), expressed in <i>E.coli</i> Arctic Express (DE3) BL 21. Lane 1: Molecular weight markers; Lanes 2-3: Supernatant and pellet, respectively, prior to Ni⁺⁺NTA column binding; Lanes 4-5: Elution fraction 1 and 2, respectively, eluted sequentially from Ni⁺⁺NTA column. B. Western blot using Penta-His antibody. Lane 1: Molecular weight markers. Lane 2: Elution fraction 2 after native purification demonstrating the expected size band (36.6 KDa). C. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of protein concentration (μg/ml). **p <0.001. D. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of time (hours). E =  elution fraction 2; AP =  alkaline phosphatase (positive control); NC =  negative control comprising supernatant obtained following IPTG induction of Arctic <i>E coli</i> transformed with empty vector. In each reaction, 1 μg/ml poly P was used as substrate. Data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-g005" target="_blank">Fig. 5C and 5D</a> are derived from individual experiments, each of which was performed three times with separate samples yielding similar results.</p

MT0516 is required for full virulence of Mtb in guinea pigs.

<p>A. Growth and survival of the mutant following low-dose aerosol infection in guinea pig lungs relative to wild-type and complement strains (*p≤0.01; **p≤0.001 for differences in bacillary counts between mutant- and wild-type-infected lungs). B. Gross pathology of Mtb-infected guinea pig lungs at Day 84 after aerosol infection. C. Microscopic examination of Mtb-infected lungs at Day 84 (H&E stain, 2× magnification. Insets: Ziehl-Neelsen staining, 50x magnification. CDC1551 =  wild-type strain; MT0516::Tn =  MT0516-deficent mutant; MT0516::Tn Comp =  MT0516::Tn complement strain.</p

Complementation of transposon mutant MT0516::Tn.

<p>A. Diagram of expected recombination between integrating plasmid and genomic DNA. B. PCR analysis of genomic DNA. A list of relevant primers is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-t001" target="_blank">Table 1</a>. Lane 1: Molecular weight markers. Lanes 2-4: Amplification of the MT0516 gene from wild-type, MT0516::Tn, and MT0516::Tn Comp, respectively. The expected 1035-bp PCR product is present in Lanes 2 and 4 but absent in Lane 3. Lane 5: Molecular weight markers. Lanes 6-8: Amplification of the kanamycin resistance cassette from wild type, MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 226-bp product only in Lanes 7 and 8. Lane 9: Molecular weight markers. Lanes 10-11: Amplification of the hygromycin resistance cassette from MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 319-bp product only in Lane 11. C. Diagram of expected sizes of <i>EcoRI</i>-digested genomic fragments expected to hybridize to probe recognizing region of MT0516 coding sequence by Southern blot (D). Note that MT0516::Tn Comp is expected to have two different size fragments hybridizing to the MT0516 probe, including that found in MT0516::Tn (1.8 Kb) and a distinct fragment unique to the attB integration site (1.7 Kb). D. Southern blot detecting the presence of DNA fragments bound to the MT0516 hybridization probe. Lane 1: Molecular weight markers. Lane 2: Blank. Lanes 3-5: Wild type, MT0516::Tn, and MT:0516::Tn Comp, respectively.</p

Protein modeling predicts that <i>M. tuberculosis</i> MT0516 is an exopolyphosphatase.

<p>The protein backbone ribbon structure was modeled by PHYRE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone.0028076-Kelley1" target="_blank">[23]</a>, showing the conserved hydrolase fold associated with exopolyphosphatases. Left-angled, top-down view of the interface canyon between domains I and II with the α helix 4 (within box) that houses the highly conserved active site E112 side chain opening into the canyon floor. The canyon walls are lined with β sheets.</p

Metrics on current versions of the BCO, ENVO, and PCO.

1<p>. For BCO and PCO, the number of relations includes only relations that point to a BCO or PCO term, to adjust for the large proportion of imported terms.</p>2<p>. 39 imported from Basic Formal Ontology, 13 imported from Information Artifact Ontology, 10 imported from Ontology for Biomedical Investigations, 1 imported from Common Anatomy Reference Ontology.</p>3<p>. 172 imported from Chemical Entities of Biological Interest, 49 from Phenotypic Quality Ontology.</p>4<p>. 39 imported from Basic Formal Ontology, 1269 imported from Gene Ontology, 11 imported from Information Artifact Ontology, 2 imported from Common Anatomy Reference Ontology.</p

Structured sampling schemes.

<p>(<b>A</b>) Biological sampling can be structured in both space and time. Environmental sampling of ocean water often includes sampling along a transect, with samples collected at multiple depths at each location. Additionally, each sample of water collected may be subsampled for metagenomic analysis or measuring chemical content. (<b>B</b>) Sampling schemes in ecological studies are often nested and may include plot; subplot or transect within plot; individual within plot, subplot, or transect; organ (e.g., leaf) within individual; tissue within organ; and DNA or mineral (e.g., C or N) within tissue. DNA extracted from a leaf of a tree that is present in a sub-plot may therefore be characterized by environmental features of the plot.</p

Core terms of the Biological Collections Ontology (BCO) and their relations to upper ontologies.

<p>Core BCO terms (in orange) are subclasses of terms from the Basic Formal Ontology (BFO – in yellow) or the Ontology for Biomedical Investigations (OBI – in blue). For example, BCO:<i>material sample</i> is a subclass of BFO:<i>material entity</i> and has role BFO:<i>material sample role</i> (which is a BFO:<i>role</i>), while BFO:<i>material sampling process</i> is a subclass of OBI:<i>planned process</i>, and has as specified output BCO:<i>material sample</i>.</p

Linking data across sites in the Genomic Observatories network's Ocean Sampling Day.

<p>(<b>A</b>) Ocean Sampling Day involves the simultaneous sampling of the world's oceans on a single day, as represented by the red stars on the map of the earth. Multiple ocean water sampling processes take place at each location. Those water samples are filtered to produce samples of organismal communities that are submitted to the bioarchive at the Smithsonian Institution. A subsample of the filtered material is analyzed to produce a metagenomic sequence, which may be stored in the Genomes Online Database (<a href="http://www.genomesonline.org/cgi-bin/GOLD/index.cgi" target="_blank">GOLD</a>). To be useful in comparative studies, data from each process at each location must be accessible and interpretable. (<b>B</b>) A graphical representation of how part of the workflow shown in <b>A</b> (from ocean water sampling to filtering to metagenomic sequencing) can be annotated with terms from multiple, coordinated ontologies and queried via an ontology-based data store. Ontology classes are shown as ovals and instances are shown as rectangles, with instances color-coded to match their parent classes. This figure shows how a metagenomic sequence and the taxa associated with it can be linked back to the original Ocean Sampling Day collecting event through a chain of inputs and outputs.</p

Linking samples and derivatives from the Moorea Biocode project.

<p>(<b>A</b>) Biodiversity data from the Moorea Biocode project were collected at many different levels that are connected to one another in biologically meaningful ways, such as an Essig Museum specimen collected as part of a Biocode bioinventory event, a tissue sample submitted to the Smithsonian Institution, a metagenomic gut sample collected from the specimen and registered with the <a href="http://camera.calit2.net/" target="_blank">CAMERA portal</a>, or DNA extracted from either the tissue or metagenomic sample. (<b>B</b>) A graphical representation of how part of the workflow shown in <b>A</b> (from field collection to tissue sampling to DNA extraction) can be annotated with terms from multiple, coordinated ontologies and queried via an ontology-based data store. Ontology classes are shown as ovals and instances are shown as rectangles, with instances color-coded to match their parent classes. This figure shows how, for example, TaxonID B resulting from the BLAST identification process on Genbank sequence B can be linked back to the original Moorea Biocode sampling process, or how a chain of inputs and outputs can be used to infer that an instance of DNA molecules is derived from an instance of an insect specimen.</p