Camilla L. Nesbø

On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales

Since publication of the first Thermotogales genome, Thermotoga maritima strain MSB8, single- and multi-gene analyses have disagreed on the phylogenetic position of this order of Bacteria. Here we present the genome sequences of 4 additional members of the Thermotogales (Tt. petrophila, Tt. lettingae, Thermosipho melanesiensis, and Fervidobacterium nodosum) and a comprehensive comparative analysis including the original T. maritima genome. While ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Firmicutes, especially Clostridia. Indeed, on the basis of the majority of genes in their genomes (including genes that are also found in Aquificales), Thermotogales should be considered members of the Firmicutes. This result highlights the conflict between the taxonomic goal of assigning every species to a unique position in an inclusive Linnaean hierarchy and the evolutionary goal of understanding phylogenesis in the presence of pervasive horizontal gene transfer (HGT) within prokaryotes. Amino acid compositions of reconstructed ancestral sequences from 423 gene families suggest an origin of this gene pool even more thermophilic than extant members of this order, followed by adaptation to lower growth temperatures within the Thermotogales.

METAGENassist: a comprehensive web server for comparative metagenomics

With recent improvements in DNA sequencing and sample extraction techniques, the quantity and quality of metagenomic data are now growing exponentially. This abundance of richly annotated metagenomic data and bacterial census information has spawned a new branch of microbiology called comparative metagenomics. Comparative metagenomics involves the comparison of bacterial populations between different environmental samples, different culture conditions or different microbial hosts. However, in order to do comparative metagenomics, one typically requires a sophisticated knowledge of multivariate statistics and/or advanced software programming skills. To make comparative metagenomics more accessible to microbiologists, we have developed a freely accessible, easy-to-use web server for comparative metagenomic analysis called METAGENassist. Users can upload their bacterial census data from a wide variety of common formats, using either amplified 16S rRNA data or shotgun metagenomic data. Metadata concerning environmental, culture, or host conditions can also be uploaded. During the data upload process, METAGENassist also performs an automated taxonomic-to-phenotypic mapping. Phenotypic information covering nearly 20 functional categories such as GC content, genome size, oxygen requirements, energy sources and preferred temperature range is automatically generated from the taxonomic input data. Using this phenotypically enriched data, users can then perform a variety of multivariate and univariate data analyses including fold change analysis, t-tests, PCA, PLS-DA, clustering and classification. To facilitate data processing, users are guided through a step-by-step analysis workflow using a variety of menus, information hyperlinks and check boxes. METAGENassist also generates colorful, publication quality tables and graphs that can be downloaded and used directly in the preparation of scientific papers. METAGENassist is available at http://www.metagenassist.ca.

Phylogeography and population history of Atlantic mackerel (Scomber scombrus L.): a genealogical approach reveals genetic structuring among the eastern Atlantic stocks

Despite the resolving power of DNA markers, pelagic and migratory marine fish species generally show very little geographical population structuring. In mackerel (Scomber scombrus L.) population differentiation has been detected only at a transatlantic scale. By applying two regions in mitochondrial DNA (mtDNA) (D–loop and cytochrome b (cytb)) in combination with genealogical and frequency–based statistical approaches, our data suggest population differentiation among eastern Atlantic spawning stocks. In contrast, and indicative of homing behaviour, no genetic structuring was observed among shoals of individuals outside the spawning season. Among spawning stocks, mtDNA D–loop sequences detected differentiation within the eastern Atlantic, while the cytb gene detected transatlantic differentiation. The impact of recurrent events (e.g. gene flow restricted by isolation by distance) and historic events (e.g. population range expansions) among spawning stocks was investigated applying a nested cladistic analysis of geographical distribution of cytb haplotype lineages. In the eastern Atlantic, historical population range expansion, presumably in connection with recolonization of northern areas after the last glaciation, is suggested to be the main factor determining mtDNA lineage distribution. This was supported by estimates of mtDNA nucleotide diversity, where the highest diversity was observed for the stock spawning in the Bay of Biscay, for which the size estimate is only 15% of the largest stock (Celtic Sea). In addition to revealing population differentiation, our data demonstrate the importance of sampling strategy and the power of applying statistical methods addressing both ongoing and historical population processes.

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.

Recombination in Thermotoga: Implications for Species Concepts and Biogeography

Here we characterize regions of the genomes of eight members of the hyperthermophilic genus Thermotoga. These bacteria differ from each other physiologically and by 3–20% in gene content and occupy physically distinct environments in widely disparate regions of the globe. Among the four different lineages (represented by nine different strains) that we compare, no two are closer than 96% in the average sequences of their genes. By most accepted recent definitions these are different “ecotypes” and different “species.” And yet we find compelling evidence for recombination between them. We suggest that no single prokaryotic species concept can accommodate such uncoupling of ecotypic and genetic aspects of cohesion and diversity, and that without a single concept, the question of whether or not prokaryotic species might in general be cosmopolitan cannot be sensibly addressed. We can, however, recast biogeographical questions in terms of the distribution of genes and their alleles.

Kosmotoga olearia gen. nov., sp. nov., a thermophilic, anaerobic heterotroph isolated from an oil production fluid

A novel thermophilic, heterotrophic bacterium, strain TBF 19.5.1(T), was isolated from oil production fluid at the Troll B oil platform in the North Sea. Cells of strain TBF 19.5.1(T) were non-motile rods with a sheath-like structure, or toga. The strain was Gram-negative and grew at 20-80 degrees C (optimum 65 degrees C), pH 5.5-8.0 (optimum pH 6.8) and NaCl concentrations of 10-60 g l(-1) (optimum 25-30 g l(-1)). For a member of the order Thermotogales, the novel isolate is capable of unprecedented growth at low temperatures, with an optimal doubling time of 175 min (specific growth rate 0.24 h(-1)) and a final optical density of >1.4 when grown on pyruvate at 37 degrees C. Various carbohydrates, proteinaceous compounds and pyruvate served as growth substrates. Thiosulfate, but not elemental sulfur, enhanced growth of the isolate. Sulfate also enhanced growth, but sulfide was not produced. The strain grew in the presence of up to approximately 15 % oxygen, but only if cysteine was included in the medium. Growth of the isolate was inhibited by acetate, lactate and propionate, while butanol and malate prevented growth. The major fermentation products formed on maltose were hydrogen, carbon dioxide and acetic acid, with traces of ethanol and propionic acid. The G+C content of the genomic DNA was 42.5 mol%. Phylogenetic analyses of the 16S and 23S rRNA gene sequences as well as 29 protein-coding ORFs placed the strain within the bacterial order Thermotogales. Based on the phylogenetic analyses and the possession of a variety of physiological characteristics not previously found in any species of this order, it is proposed that the strain represents a novel species of a new genus within the family Thermotogaceae, order Thermotogales. The name Kosmotoga olearia gen. nov., sp. nov. is proposed. The type strain of Kosmotoga olearia is TBF 19.5.1(T) (=DSM 21960(T) =ATCC BAA-1733(T)).

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.

Suppressive Subtractive Hybridization Detects Extensive Genomic Diversity in Thermotoga maritima

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined Thermotoga maritima, a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of T. maritima MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from Thermotoga sp. strain RQ2, a very close relative (approximately 96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order Thermotogales, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.

Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of approximately 3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling (Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.

Evidence for existence of "mesotogas," members of the order Thermotogales adapted to low-temperature environments.

ABSTRACT All cultivated isolates of the bacterial order Thermotogales are either thermophiles or hyperthermophiles, but Thermotogales 16S rRNA gene sequences have been detected in many mesophilic anaerobic and microaerophilic environments, particularly within communities involved in the remediation of pollutants. Here we provide metagenomic evidence for the existence of Thermotogales lineages, which we informally call “mesotoga,” that are adapted to growth at lower temperatures. Two fosmid clones containing mesotoga DNA, originating from a low-temperature enrichment culture that degrades a polychlorinated biphenyl congener, were sequenced. Phylogenetic analysis clearly puts this bacterial lineage within the Thermotogales order, with the rRNA gene trees and 21 of 58 open reading frames strongly supporting this relationship. An analysis of protein sequence composition showed that mesotoga proteins are adapted to function at lower temperatures than are their identifiable homologs from thermophilic and hyperthermophilic members of the order Thermotogales, supporting the notion that this bacterium lives and grows optimally at lower temperatures. The phylogenetic analysis suggests that the mesotoga lineage from which our fosmids derive has used both the acquisition of genes from its neighbors and the modification of existing thermophilic sequences to adapt to a mesophilic lifestyle.