Stuart P. Donachie

Microbial community structure in the North Pacific ocean.

We report a ribosomal tag pyrosequencing study of the phylogenetic diversity of Archaea, Bacteria and Eucarya over a depth profile at the Hawaii Ocean Time-Series Station, ALOHA. The V9 region of the SSU rRNA gene was amplified from samples representing the epi- (10 m), meso- (800 m) and bathy- (4400 m) pelagia. The primers used are expected to amplify representatives of ∼80% of known phylogenetic diversity across all three domains. Comparisons of unique sequences revealed a remarkably low degree of overlap between communities at each depth. The 444 147 sequence tags analyzed represented 62 975 unique sequences. Of these, 3707 (5.9%) occurred at two depths, and only 298 (0.5%) were observed at all three depths. At this level of phylogenetic resolution, Bacteria diversity decreased with depth but was still equivalent to that reported elsewhere for different soil types. Archaea diversity was highest in the two deeper samples. Eucarya observations and richness estimates are almost one order of magnitude higher than any previous marine microbial Eucarya richness estimates. The associations of many Eucarya sequences with putative parasitic organisms may have significant impacts on our understanding of the mechanisms controlling host population density and diversity, and point to a more significant role for microbial Eucarya in carbon flux through the microbial loop. We posit that the majority of sequences detected from the deep sea that have closest matches to sequences from non-pelagic sources are indeed native to the marine environment, and are possibly responsible for key metabolic processes in global biogeochemical cycles.

Genome sequence of the deep-sea γ-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy

We report the complete genome sequence of the deep-sea γ-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Lōihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other γ-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Culture clash: challenging the dogma of microbial diversity

The advent of ribosomal RNA sequence analysis 30 years ago revolutionized microbial ecology and significantly expanded our knowledge of microbial phylogenetic diversity (Woese and Fox, 1977). However, exclusive reliance on this single approach in environmental studies perpetuates certain assumptions that should be discussed and re-evaluated. These assumptions include (1) ribosomal RNA gene analyses can be used exclusively for the study of microbial diversity; (2) the number of colonyforming units (CFU) on nutrient media as a fraction of the total number of bacteria determined by microscopy is related to phylogenetic diversity and (3) only a fraction of environmental bacteria ‘species’ are culturable. Overlooking a century of cultivation history and encouraging use only of ribosomal approaches leads to significant gaps in microbial community diversity data. We demonstrate that cultivation methods are critical in microbial diversity studies and that they detect organisms undetected by molecular techniques. We thus caution against single-method approaches and posit that metagenomic techniques may be complemented by parallel culture libraries whenever diversity information is sought.

Transcript amplification from single bacterium for transcriptome analysis

Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87-0.89) and drop-outs (4%-6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.

The Hawaiian Archipelago: A Microbial Diversity Hotspot

The Hawaiian Archipelago is a “biodiversity hotspot” where significant endemism among eukaryotes has evolved through geographic isolation and local topography. To address the absence of corresponding region-wide data on Hawaii’s microbiota, we compiled the first 16S SSU rDNA clone libraries and cultivated bacteria from five Hawaiian lakes, an anchialine pool, and the Lō’ihi submarine volcano. These sites offer diverse niches over ~5000 m elevation and ~1150 nautical miles. Each site hosted a distinct prokaryotic community dominated by Bacteria. Cloned sequences fell into 158 groups from 18 Bacteria phyla, while seven were unassigned and two belonged in the Euryarchaeota. Only seven operational taxonomic units (each OTU comprised sequences that shared ≥97% sequence identity) occurred in more than one site. Pure bacterial cultures from all sites fell into 155 groups (each group comprised pure cultures that shared ≥97% 16S SSU rDNA sequence identity) from 10 Bacteria phyla; 15 Proteobacteria and Firmicutes were cultivated from more than one site. One hundred OTUs (60%) and 52 (33.3%) cultures shared <97% 16S SSU rDNA sequence identity with published sequences. Community structure reflected habitat chemistry; most δ-Proteobacteria occurred in anoxic and sulfidic waters of one lake, while β-Proteobacteria were cultivated exclusively from fresh or brackish waters. Novel sequences that affiliate with an Antarctic-specific clade of Deinococci, and Candidate Divisions TM7 and BRC1, extend the geographic ranges of these phyla. Globally and locally remote, as well as physically and chemically diverse, Hawaiian aquatic habitats provide unique niches for the evolution of novel communities and microorganisms.

Phylogenomics Reveals Convergent Evolution of Lifestyles in Close Relatives of Animals and Fungi.

The Opisthokonta are a eukaryotic supergroup divided in two main lineages: animals and related protistan taxa, and fungi and their allies [1, 2]. There is a great diversity of lifestyles and morphologies among unicellular opisthokonts, from free-living phagotrophic flagellated bacterivores and filopodiated amoebas to cell-walled osmotrophic parasites and saprotrophs. However, these characteristics do not group into monophyletic assemblages, suggesting rampant convergent evolution within Opisthokonta. To test this hypothesis, we assembled a new phylogenomic dataset via sequencing 12 new strains of protists. Phylogenetic relationships among opisthokonts revealed independent origins of filopodiated amoebas in two lineages, one related to fungi and the other to animals. Moreover, we observed that specialized osmotrophic lifestyles evolved independently in fungi and protistan relatives of animals, indicating convergent evolution. We therefore analyzed the evolution of two key fungal characters in Opisthokonta, the flagellum and chitin synthases. Comparative analyses of the flagellar toolkit showed a previously unnoticed flagellar apparatus in two close relatives of animals, the filasterean Ministeria vibrans and Corallochytrium limacisporum. This implies that at least four different opisthokont lineages secondarily underwent flagellar simplification. Analysis of the evolutionary history of chitin synthases revealed significant expansions in both animals and fungi, and also in the Ichthyosporea and C. limacisporum, a group of cell-walled animal relatives. This indicates that the last opisthokont common ancestor had a complex toolkit of chitin synthases that was differentially retained in extant lineages. Thus, our data provide evidence for convergent evolution of specialized lifestyles in close relatives of animals and fungi from a generalist ancestor.

Bacteria in the sea-ice zone between Elephant Island and the South Orkneys during the Polish sea-ice zone expedition, (December 1988 to January 1989)

During austral summer 1988/89, total bacterial Acridine Orange Direct Counts (AODC) in seawater, mean 6.0×106 l−1, were three to ten times lower than generally reported for the Bransfield Strait to north Weddell Sea area. In contrast, numbers of viable bacteria (Colony Forming Units, CFU), mean 10.6×103 l−1 were two to three times higher than reported. Bacterial abundance here shows large seasonal and spatial changes. On the basis of bacterial, diatom, detritus, and amino acid data from this study, two main regions were defined: ‘Cold winter water’ in the west with high salinity and low CFU, AODC, and other parameters. In the east, lower salinity and higher values for all parameters were found in warmer meltwater at the surface. CFU and AODC values in ice were respectively six and 85 times higher than in surrounding seawater. Taxonomic studies indicate considerable diversity in genera and nutritional requirements of isolated bacteria. Sea-ice and water column bacterial communities differed. Many isolated strains, however, were found in both habitats. Sea-ice seems to be important in regulating surrounding bacterioplankton.

Bacterial digestive enzyme activity in the stomach and hepatopancreas of Meganyctiphanes norvegica (M. Sars, 1857).

Northern krill, Meganyctiphaunes norvegica were maintained in antibiotic-treated (“A”), and untreated (control, “C”) aquaria. Each 24 h (for 72 h) numbers of saprophytic (Colony Forming Units, CFU), chitinolytic, and total bacteria (Acridine Orange Direct Count, AODC), were determined in the stomachs and hepatopancreata of animals. Parallely, the activities of chitinase, N-acetyl-β-d-glucosaminidase (NAGase), protease, cellulase and laminarinase were measured, and bacterial and endogenous chitinases compared by Fast Protein Liquid Chromatography (FPLC). CFU in “C” group stomachs were stable, but varied widely in the “A” group. AODC did not vary to any great extent. Hepatopancreas CFU decreased by four orders in the “A” compared to the “C” group. In “C” hepatopancreata, AODC were slighly higher than in “A”. Numbers of chitinolytic bacteria fell steeply in the “A” group. Chitinase, NAGase, protease, and cellulase activities in the stomach were significantly lower (5% level) after 24 h in “A” than in “C”. Laminarinase activity was significantly reduced in both organs after 72 h. Hepatopancreas chitinase and protease activities showed significant reductions only after 24 h. FPLC elution profiles of NAGase from krill, and bacteria grown on a medium semi-selective for chitinolytic bacteria differed. A number of bacterial chitinase peaks were absent in the “A” group. The API Zym system showed reduced proteolytic and lipase (C14) activity over 72 h. Endogenous bacteria are considered commensal with respect to the enzymes discussed.

Dynamics of genomic innovation in the unicellular ancestry of animals

Which genomic innovations underpinned the origin of multicellular animals is still an open debate. Here, we investigate this question by reconstructing the genome architecture and gene family diversity of ancestral premetazoans, aiming to date the emergence of animal-like traits. Our comparative analysis involves genomes from animals and their closest unicellular relatives (the Holozoa), including four new genomes: three Ichthyosporea and Corallochytrium limacisporum. Here, we show that the earliest animals were shaped by dynamic changes in genome architecture before the emergence of multicellularity: an early burst of gene diversity in the ancestor of Holozoa, enriched in transcription factors and cell adhesion machinery, was followed by multiple and differently-timed episodes of synteny disruption, intron gain and genome expansions. Thus, the foundations of animal genome architecture were laid before the origin of complex multicellularity – highlighting the necessity of a unicellular perspective to understand early animal evolution. DOI: http://dx.doi.org/10.7554/eLife.26036.001

Spatial transcriptomes within the Pseudomonas aeruginosa biofilm architecture: P. aeruginosa spatial transcriptomes

Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene‐expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm‐solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.