Tim J. Dumonceaux

The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis

Flowering plants display a remarkable range of inflorescence architecture, and pedicel characteristics are one of the key contributors to this diversity. However, very little is known about the genes or the pathways that regulate pedicel development. The brevipedicellus (bp) mutant of Arabidopsis thaliana displays a unique phenotype with defects in pedicel development causing downward-pointing flowers and a compact inflorescence architecture. Cloning and molecular analysis of two independent mutant alleles revealed that BP encodes the homeodomain protein KNAT1, a member of the KNOX family. bp-1 is a null allele with deletion of the entire locus, whereas bp-2 has a point mutation that is predicted to result in a truncated protein. In both bp alleles, the pedicels and internodes were compact because of fewer cell divisions; in addition, defects in epidermal and cortical cell differentiation and elongation were found in the affected regions. The downward-pointing pedicels were produced by an asymmetric effect of the bp mutation on the abaxial vs. adaxial sides. Cell differentiation, elongation, and growth were affected more severely on the abaxial than adaxial side, causing the change in the pedicel growth angle. In addition, bp plants displayed defects in cell differentiation and radial growth of the style. Our results show that BP plays a key regulatory role in defining important aspects of the growth and cell differentiation of the inflorescence stem, pedicel, and style in Arabidopsis.

Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken.

ABSTRACT The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.

Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-Based Diets by Chaperonin-60 Sequencing and Quantitative PCR

ABSTRACT We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition.

ABSTRACT We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances (“barcode gap”) was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Detection of polyoma and corona viruses in bats of Canada.

Several instances of emerging diseases in humans appear to be caused by the spillover of viruses endemic to bats, either directly or through other animal intermediaries. The objective of this study was to detect, identify and characterize viruses in bats in the province of Manitoba and other regions of Canada. Bats were sampled from three sources: live-trapped Myotis lucifugus from Manitoba, rabies-negative Eptesicus fuscus, M. lucifugus, M. yumanensis, M. septentrionalis, M. californicus, M. evotis, Lasionycteris (L.) noctivagans and Lasiurus (Las.) cinereus, provided by the Centre of Expertise for Rabies of the Canadian Food Inspection Agency (CFIA), and L. noctivagans, Las. cinereus and Las. borealis collected from a wind farm in Manitoba. We attempted to isolate viruses from fresh tissue samples taken from trapped bats in cultured cells of bat, primate, rodent, porcine, ovine and avian origin. We also screened bat tissues by PCR using primers designed to amplify nucleic acids from members of certain families of viruses. We detected RNA of a group 1 coronavirus from M. lucifugus (3 of 31 animals) and DNA from an as-yet undescribed polyomavirus from female M. lucifugus (4 of 31 animals) and M. californicus (pooled tissues from two females).

Cellobiose dehydrogenase is essential for wood invasion and nonessential for kraft pulp delignification by Trametes versicolor

Abstract Cellobiose dehydrogenase (CDH)-deficient strains of the basidiomycete Trametes versicolor were produced by transforming protoplasts of strain 52J with a plasmid carrying the T. versicolor cdh gene disrupted with a phleomycin resistance cassette. Of 143 phleomycin-resistant colonies analyzed, 3 did not produce measurable CDH during two successive two-week culture periods. Two of these mutants were shown to lack functional CDH when grown in CDH induction medium. They biobleached and delignified industrial unbleached kraft pulp as efficiently as did wild-type T. versicolor, indicating that CDH is not required for the degradation and biobleaching of kraft lignin. The ability to degrade 14C-guaiacyl dehydrogenative polymerizate (synthetic lignin) also appeared to be unaffected. However, compared to the parent strain, all three mutants grew poorly on minimal agar with highly crystalline cellulose as the sole carbon source. This difference was not observed on non-crystalline carbohydrates. All three mutants had a greatly decreased ability to colonize and degrade both seasoned and fresh native white birch wood, a natural substrate of T. versicolor. The dramatic decrease in T. versicolor 52J’s ability to invade and grow on birch wood caused by the loss of its secreted CDH strongly suggests that this enzyme is essential to its wood invading and degrading niche in the forest ecosystem.

CLONING AND SEQUENCING OF A GENE ENCODING CELLOBIOSE DEHYDROGENASE FROM TRAMETES VERSICOLOR

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellulose-degrading conditions by Trametes versicolor strain 52J (Tv) and several other wood-degrading fungi, including Phanerochaete chrysosporium (Pc). In order to understand better the nature and properties of this enzyme, we isolated a genomic clone of Tv cdh using heterologous probes derived from the sequence of Pc cdh. DNA sequence analysis revealed that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a single copy in the strain of Tv analyzed. Tv cdh was shown by Northern blot analysis to be expressed as a single transcript under cellulolytic conditions. RT-PCR of poly(A)+ RNA isolated under cellulolytic conditions was used to generate a near full-length cDNA copy of the cdh mRNA. The deduced protein encoded by Tv cdh consists of 768 amino acids (aa), including a predicted 19 aa signal peptide. The protein had 73% identity to the corresponding protein from Pc, which is the only other CDH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothesized to function in heme binding; the C-terminal flavin domain contained a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homology to bacterial or fungal cellulose binding domains was observed. However, a strong homology was observed to a region of Pc CDH that is hypothesized to be involved in cellulose binding.

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.

Molecular definition of vaginal microbiota in East African commercial sex workers.

ABSTRACT Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV+) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV+ individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV+ women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV+ and BV− diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV+ and BV−. Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV− (“normal”) phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.