Jonathan L. Klassen

Microbial Strain Prioritization Using Metabolomics Tools for the Discovery of Natural Products

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Getting the Hologenome Concept Right: an Eco-Evolutionary Framework for Hosts and Their Microbiomes

Given the complexity of host-microbiota symbioses, scientists and philosophers are asking questions at new biological levels of hierarchical organization—what is a holobiont and hologenome? When should this vocabulary be applied? Are these concepts a null hypothesis for host-microbe systems or limited to a certain spectrum of symbiotic interactions such as host-microbial coevolution? Critical discourse is necessary in this nascent area, but productive discourse requires that skeptics and proponents use the same lexicon. ABSTRACT Given the complexity of host-microbiota symbioses, scientists and philosophers are asking questions at new biological levels of hierarchical organization—what is a holobiont and hologenome? When should this vocabulary be applied? Are these concepts a null hypothesis for host-microbe systems or limited to a certain spectrum of symbiotic interactions such as host-microbial coevolution? Critical discourse is necessary in this nascent area, but productive discourse requires that skeptics and proponents use the same lexicon. For instance, critiquing the hologenome concept is not synonymous with critiquing coevolution, and arguing that an entity is not a primary unit of selection dismisses the fact that the hologenome concept has always embraced multilevel selection. Holobionts and hologenomes are incontrovertible, multipartite entities that result from ecological, evolutionary, and genetic processes at various levels. They are not restricted to one special process but constitute a wider vocabulary and framework for host biology in light of the microbiome.

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.

Metagenomics of Hydrocarbon Resource Environments Indicates Aerobic Taxa and Genes to be Unexpectedly Common

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

Microbial Communities Involved in Methane Production from Hydrocarbons in Oil Sands Tailings

Microbial metabolism of residual hydrocarbons, primarily short-chain n-alkanes and certain monoaromatic hydrocarbons, in oil sands tailings ponds produces large volumes of CH(4) in situ. We characterized the microbial communities involved in methanogenic biodegradation of whole naphtha (a bitumen extraction solvent) and its short-chain n-alkane (C(6)-C(10)) and BTEX (benzene, toluene, ethylbenzene, and xylenes) components using primary enrichment cultures derived from oil sands tailings. Clone libraries of bacterial 16S rRNA genes amplified from these enrichments showed increased proportions of two orders of Bacteria: Clostridiales and Syntrophobacterales, with Desulfotomaculum and Syntrophus/Smithella as the closest named relatives, respectively. In parallel archaeal clone libraries, sequences affiliated with cultivated acetoclastic methanogens (Methanosaetaceae) were enriched in cultures amended with n-alkanes, whereas hydrogenotrophic methanogens (Methanomicrobiales) were enriched with BTEX. Naphtha-amended cultures harbored a blend of these two archaeal communities. The results imply syntrophic oxidation of hydrocarbons in oil sands tailings, with the activities of different carbon flow pathways to CH(4) being influenced by the primary hydrocarbon substrate. These results have implications for predicting greenhouse gas emissions from oil sands tailings repositories.

Microtermolides A and B from Termite-Associated Streptomyces sp. and Structural Revision of Vinylamycin

Microtermolides A (1) and B (2) were isolated from a Streptomyces sp. strain associated with fungus-growing termites. The structures of 1 and 2 were determined by 1D- and 2D-NMR spectroscopy and high-resolution mass spectrometry. Structural elucidation of 1 led to the re-examination of the structure originally proposed for vinylamycin (3). Based on a comparison of predicted and experimental 1H and 13C NMR chemical shifts, we propose that vinylamycin’s structure be revised from 3 to 4.

Bacterial diversity associated with ornithogenic soil of the Ross Sea region, Antarctica.

In the Ross Sea region of Antarctica, ornithogenic soils form on land under Adélie Penguin rookeries. Compared with mineral soils of the Ross Sea region, ornithogenic soils are generally high in microbial biomass, organic carbon, and total nitrogen and phosphorus, with high electrical conductivity and large variations in pH. The objective of this study was to assess the bacterial composition of ornithogenic soils from Cape Hallett and Cape Bird in the Ross Sea region using culture-independent methods. Soil clone libraries were constructed and those clones that occurred > or = 3 times were sequenced. The bacterial diversity of the soils was dependent on the presence of penguins. Firmicutes most closely related to the endospore-formers (e.g., Oceanobacillus profundus and Clostridium acidurici) and (or) Gammaproteobacteria belonging to the genus Psychrobacter dominated soils currently occupied with penguins. In contrast, Gammaproteobacteria, closely related to cultured members of the genera Rhodanobacter, Psychrobacter, Dokdonella, and Lysobacter, dominated the soils previously colonized by penguins. Results of this study indicate that despite relatively high nutrient levels and microbial biomass, bacterial communities of ornithogenic soils were not more diverse than those of mineral soils of the Ross Sea region of Antarctica.

Characterization of Hymenobacter isolates from Victoria Upper Glacier, Antarctica reveals five new species and substantial non-vertical evolution within this genus

We isolated several Hymenobacter-like strains from Victoria Upper Glacier, Antarctica, basal ice that diverged substantially from currently defined Hymenobacter species according to their 16S rRNA and gyrB gene phylogenies. All strains were psychrotolerant, heterotrophic aerobes which grew preferentially on low salt and low nutrient strength agar. Further phenotypic and chemotaxonomic characterization of these isolates supported their assignment as five novel species: H. algoricola sp. nov., H. antarcticus sp. nov., H. elongatus sp. nov., H. fastidiosus sp. nov., and H. glaciei sp. nov. Remarkable among these data was the prevalence of horizontal gene transfers and phenotypic variation, even between apparently closely related strains. These results suggest extensive non-vertical evolution within the genus Hymenobacter, and may reflect evolutionary trajectories resulting from dormancy, e.g., during interment in glacial ice.

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

Background Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data. Methodology/Principal Findings Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection. Conclusions/Significance Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a “bramble” model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic “root”. Structural diversification may be constrained (“trimmed”) where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification.

Differences in Carotenoid Composition among Hymenobacter and Related Strains Support a Tree-Like Model of Carotenoid Evolution

ABSTRACT Carotenoids are structurally diverse pigments of biotechnological interest as natural colorants and in the prevention of human disease. The carotenoids present in 19 strains taxonomically related to the poorly described, nonphotosynthetic bacterial genus Hymenobacter, including 10 novel isolates cultivated from Victoria Upper Glacier, Antarctica, were characterized using high-performance liquid chromatography (HPLC). Nine chemically distinct carotenoids, present in various combinations irresolvable by conventional crude spectrophotometric analyses, were purified by preparative HPLC and characterized using UV-visible light absorption spectroscopy and high-resolution mass spectrometry. All major Hymenobacter carotenoids appear to be derived from a common backbone of 2′-hydroxyflexixanthin and include previously unreported presumptive hexosyl, pentosyl, and methyl derivatives. Their distribution does not, however, correlate perfectly with 16S rRNA gene phylogeny. Carotenoid composition, therefore, may be strain specific and does not follow a strictly homogeneous pattern of vertical evolutionary descent.