Derrick A. Horne, Composition

Invictus (from the poem by William Ernest Henley); Frantic; Clatter; *Note: Guitar ensemble work not performe

Derrick A. Horne, Composition

Invictus (from the poem by William Ernest Henley); Frantic; Clatter; *Note: Guitar ensemble work not performe

In Situ Cultured Bacterial Diversity from Iron Curtain Cave, Chilliwack, British Columbia, Canada

The culturable bacterial diversity from Iron Curtain Cave, Chilliwack, British Columbia, Canada was examined. Sixty five bacterial isolates were successfully cultivated, purified, and identified based on 16S rRNA gene sequencing. Four distinguishable phyla, i.e., Actinobacteria (44.61%), Proteobacteria (27.69%), Firmicutes (20%) and Bacteroidetes (7.69%) were identified. Arthrobacter (21.53%) was identified as the major genus, followed by Sporosarcina (9.23%), Stenotrophomonas (9.23%), Streptomyces (6.15%), Brevundimonas (4.61%), and Crocebacterium (2.8%). Noteworthy, 12.3% of the population was recognized as unidentified bacteria. The isolates were evaluated for their potential antimicrobial activities against multidrug resistant microbial strains. Two species of the genus Streptomyces exhibited a wide range of antimicrobial activities against multidrug resistance (MDR) strains of Escherichia coli and Pseudomonas spp. along with non-resistant strains of Staphylococcus aureus and E. coli. However, all of the antimicrobial activities were only observed when the isolates were grown at 8 °C in different media. To the best of our knowledge, this is the first study conducted on the Iron Curtain Cave’s bacterial diversity, and reveals some bacterial isolates that have never been reported from a cave. Bacterial isolates identified with antimicrobial properties demonstrated that the Iron Curtain Cave can be further considered as a potential habitat for antimicrobial agents.Other UBCNon UBCReviewedFacult

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores

Cure from the cave: volcanic cave actinomycetes and their potential in drug discovery

Volcanic caves have been little studied for their potential as sources of novel microbial species and bioactive compounds with new scaffolds. We present the first study of volcanic cave microbiology from Canada and suggest that this habitat has great potential for the isolation of novel bioactive substances. Sample locations were plot ted on a contour map that was compiled in ArcView 3.2. Over 400 bacterial isolates were obtained from the Helmcken Falls cave in Wells Gray Provincial Park, British Columbia. From our preliminary screen, of 400 isolates tested, 1% showed activity against extended spectrum ß-lactamase E. coli, 1.75% against Escherichia coli, 2.25% against Acinetobacter baumannii, and 26.50% against Klebsiella pneumoniae. In addition, 10.25% showed activity against Micrococcus luteus, 2% against methicillin resistant Staphylococcus aureus, 9.25% against Mycobacterium smegmatis, 6.25% Pseudomonas aeruginosa and 7.5% against Candida albicans. Chemical and physical characteristics of three rock wall samples were studied using scanning electron microscopy and f lame atomic absorption spectrometry. Calcium (Ca), iron (Fe), and aluminum (Al) were the most abundant components while magnesium (Mg), sodium (Na), arsenic (As), lead (Pb), chromium (Cr), and barium (Ba) were second most abundant with cadmium (Cd) and potassium (K) were the least abundant in our samples. Scanning electron microscopy (SEM) showed the presence of microscopic life forms in all three rock wall samples. 16S rRNA gene sequencing of 82 isolates revealed that 65 (79.3%) of the strains belong to the Streptomyces genus and 5 (6.1%) were members of Bacillus, Pseudomonas, Nocardia and Erwinia genera. Interestingly, twelve (14.6%) of the 16S rRNA sequences showed similarity to unidentified ribosomal RNA sequences in the library databases, the sequences of these isolates need to be further investigated using the EzTaxon-e database (http://eztaxon-e. ezbiocloud.net/) to determine whether or not these are novel species. Nevertheless, this suggests the possibility that they could be unstudied or rare bacteria. The Helmcken Falls cave microbiome possesses a great diversity of microbes with the potential for studies of novel microbial interactions and the isolation of new types of antimicrobial agents

Strategy for targeted knockout of <i>PpASCL</i> and genotyping of the resulting stable transformants by PCR.

<p>(a) Schematic diagram of insertion of the linear knockout construct into the <i>PpASCL</i> locus via double homologous recombination. 35S-P, CaMV 35S promoter; <i>nptII</i>, neomycin phosphotransferase II gene; 35S-T, CaMV 35S transcription termination signal. (b) Schematic diagram of recombined gene locus after successful insertion. Expected PCR product sizes based on sequence information are shown. Single-headed arrows denote the locations of primers specific to <i>PpASCL</i> (Primers 1 and 2, which bind to genomic DNA sequences located outside the locus-specific regions used for homologous recombination) or to the <i>nptII</i> resistance cassette (Primers 3 and 4) used in the PCR analyses. Primer 1, ASCL-gDNA-F; 2, ASCL-gDNA-R; 3, pTN182-5ʹ-R; 4, pTN182-3ʹ-F. Primer sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146817#pone.0146817.s005" target="_blank">S1 Table</a>. (c) PCR products using locus-specific primers 1 and 2 with DNA from untransformed control and each of three stable putative <i>PpASCL</i> knockout lines: <i>ascl-1</i>, <i>-2</i> and <i>-3</i>. (d) PCR products, indicative of 5′ and 3′ recombination between the knockout vector and homologous DNA in the <i>PpASCL</i> locus, using primers 1 plus 3 (5ʹ recombination) and primers 2 plus 4 (3ʹ recombination). Amplified DNA products were resolved electrophoretically on 1.2% agarose gels and visualized by ethidium bromide fluorescence.</p

Developmental timelines for <i>pabB4</i>, the untransformed control strain, and <i>ascl-2</i> sporophytes and spores.

<p>Photomicrographs of the typical morphologies of sporophytes and spores of control (A–N) and <i>ascl-2</i> (a–p) at successive developmental stages. The number of days after irrigation of the cultures is shown with the names assigned to each sporophytic stage. Some stages have been subdivided to allow more detailed description of changes in spore development. White arrowheads denote the outlines of the spore masses within capsules. No spores are seen during the initial growth and expanding capsule stages. The control did not reach the brown sporophytic stage during the observation period. Sporophyte scale bars = 500 μm; Spore scale bars = 10 μm.</p

Reaction sequence for the biosynthesis of hydroxylated alkylpyrones as sporopollenin building blocks.

<p>Medium- to long-chain fatty acids are produced in plastids and then translocated out to be used for the consecutive action of enzymes in sporopollenin biosynthesis. This proposed pathway produces sporopollenin building blocks that are polymerized along with fatty alcohols and phenylpropanoid acids on the surface of the spore or pollen wall by the formation of ester and ether linkages. Enzymes are listed to the right of the arrows with their corresponding reactions on the left. ACOS, acyl-CoA synthetase; ASCL, anther-specific chalcone synthase-like enzyme; MS2, Male Sterility 2; TKPR, tetraketide α-pyrone reductase.</p

Transmission electron micrographs of <i>pabB4</i> control and ascl-2 spores.

<p>Cross sections of spores from mature orange control (a) and <i>ascl-2</i> (b) sporophytes were examined with TEM. An amorphous layer found below much of the perine is indicated by an arrow in (b). Ex, exine; In, intine; Pe, perine; PM, plasma membrane. Scale bars = 500 nm.</p

Photomicrographs of cryosectioned <i>pabB4</i> control and <i>ascl-2</i> sporophytes.

<p>Cross sections of control (20 μm, a–e) and <i>ascl-2</i> (30 μm, f–j) sporophytes were taken at the yellow (a,f) and orange (b–e, g–j) stages. Images were saved before (a,b,f,g) and after toluidine blue O staining (c–e, h–j). Orbicules present in locules of the air-space and on the tapetum wall surface are indicated with red arrows in (e) and (j). Sections (e) and (j) are magnified images of red-boxed areas of (d) and (i), respectively. Co, columella; E, epidermis; Lo, locule; T, tapetum. Scale bars = 100 μm.</p