Host hybridization as a potential mechanism of lateral symbiont transfer in deep-sea vesicomyid clams.

Deep-sea vesicomyid clams live in mutualistic symbiosis with chemosynthetic bacteria that are inherited through the maternal germ line. On evolutionary timescales, strictly vertical transmission should lead to cospeciation of host mitochondrial and symbiont lineages; nonetheless, examples of incongruent phylogenies have been reported, suggesting that symbionts are occasionally horizontally transmitted between host species. The current paradigm for vesicomyid clams holds that direct transfers cause host shifts or mixtures of symbionts. An alternative hypothesis suggests that hybridization between host species might explain symbiont transfers. Two clam species, Archivesica gigas and Phreagena soyoae, frequently co-occur at deep-sea hydrocarbon seeps in the eastern Pacific Ocean. Although the two species typically host gammaproteobacterial symbiont lineages marked by divergent 16S rRNA phylotypes, we identified a number of clams with the A. gigas mitotype that hosted symbionts with the P. soyoae phylotype. Demographic inference models based on genome-wide SNP data and three Sanger sequenced gene markers provided evidence that A. gigas and P. soyoae hybridized in the past, supporting the hypothesis that hybridization might be a viable mechanism of interspecific symbiont transfer. These findings provide new perspectives on the evolution of vertically transmitted symbionts and their hosts in deep-sea chemosynthetic environments.

Pattern and synchrony of gene expression among sympatric marine microbial populations.

Planktonic marine microbes live in dynamic habitats that demand rapid sensing and response to periodic as well as stochastic environmental change. The kinetics, regularity, and specificity of microbial responses in situ, however, are not well-described. We report here simultaneous multitaxon genome-wide transcriptome profiling in a naturally occurring picoplankton community. An in situ robotic sampler using a Lagrangian sampling strategy enabled continuous tracking and repeated sampling of coherent microbial populations over 2 d. Subsequent RNA sequencing analyses yielded genome-wide transcriptome profiles of eukaryotic (Ostreococcus) and bacterial (Synechococcus) photosynthetic picoplankton as well as proteorhodopsin-containing heterotrophs, including Pelagibacter, SAR86-cluster Gammaproteobacteria, and marine Euryarchaea. The photosynthetic picoplankton exhibited strong diel rhythms over thousands of gene transcripts that were remarkably consistent with diel cycling observed in laboratory pure cultures. In contrast, the heterotrophs did not cycle diurnally. Instead, heterotrophic picoplankton populations exhibited cross-species synchronous, tightly regulated, temporally variable patterns of gene expression for many genes, particularly those genes associated with growth and nutrient acquisition. This multitaxon, population-wide gene regulation seemed to reflect sporadic, short-term, reversible responses to high-frequency environmental variability. Although the timing of the environmental responses among different heterotrophic species seemed synchronous, the specific metabolic genes that were expressed varied from taxon to taxon. In aggregate, these results provide insights into the kinetics, diversity, and functional patterns of microbial community response to environmental change. Our results also suggest a means by which complex multispecies metabolic processes could be coordinated, facilitating the regulation of matter and energy processing in a dynamically changing environment.

Microbial community transcriptomes reveal microbes and metabolic pathways associated with dissolved organic matter turnover in the sea.

Marine dissolved organic matter (DOM) contains as much carbon as the Earth's atmosphere, and represents a critical component of the global carbon cycle. To better define microbial processes and activities associated with marine DOM cycling, we analyzed genomic and transcriptional responses of microbial communities to high-molecular-weight DOM (HMWDOM) addition. The cell density in the unamended control remained constant, with very few transcript categories exhibiting significant differences over time. In contrast, the DOM-amended microcosm doubled in cell numbers over 27 h, and a variety of HMWDOM-stimulated transcripts from different taxa were observed at all time points measured relative to the control. Transcripts significantly enriched in the HMWDOM treatment included those associated with two-component sensor systems, phosphate and nitrogen assimilation, chemotaxis, and motility. Transcripts from Idiomarina and Alteromonas spp., the most highly represented taxa at the early time points, included those encoding TonB-associated transporters, nitrogen assimilation genes, fatty acid catabolism genes, and TCA cycle enzymes. At the final time point, Methylophaga rRNA and non-rRNA transcripts dominated the HMWDOM-amended microcosm, and included gene transcripts associated with both assimilatory and dissimilatory single-carbon compound utilization. The data indicated specific resource partitioning of DOM by different bacterial species, which results in a temporal succession of taxa, metabolic pathways, and chemical transformations associated with HMWDOM turnover. These findings suggest that coordinated, cooperative activities of a variety of bacterial "specialists" may be critical in the cycling of marine DOM, emphasizing the importance of microbial community dynamics in the global carbon cycle.

Evidence for homologous recombination in intracellular chemosynthetic clam symbionts.

Homologous recombination is a fundamental mechanism for the genetic diversification of free-living bacteria. However, recombination may be limited in endosymbiotic bacteria, as these taxa are locked into an intracellular niche and may rarely encounter sources of foreign DNA. This study tested the hypothesis that vertically transmitted endosymbionts of deep-sea clams (Bivalvia: Vesicomyidae) show little or no evidence of recombination. Phylogenetic analysis of 13 loci distributed across the genomes of 14 vesicomyid symbionts revealed multiple, well-supported inconsistencies among gene tree topologies, and maximum likelihood-based tests rejected a hypothesis of shared evolutionary history (linkage) among loci. Further, multiple statistical methods confirmed the presence of recombination by detecting intragenic breakpoints in two symbiont loci. Recombination may be confined to a subset of vesicomyid symbionts, as some clades showed high levels of genomic stability, whereas others showed clear patterns of homologous exchange. Notably, a mosaic genome is present in symB, a symbiont lineage shown to have been acquired laterally (i.e., nonvertically) by Vesicomya sp. JdF clams. The majority of loci analyzed here supported a tight sister clustering of symB with the symbiont of a host species from the Mid-Atlantic Ridge, whereas others placed symB in a clade with symA, the dominant phylotype of V. sp. JdF clams. This result raises the hypothesis that lateral symbiont transfer between hosts may facilitate recombination by bringing divergent symbiont lineages into contact. Together, the data show that homologous recombination contributes to the diversification of vesicomyid clam symbionts, despite the intracellular lifestyle of these bacteria.

Lateral symbiont acquisition in a maternally transmitted chemosynthetic clam endosymbiosis.

Deep-sea clams of the family Vesicomyidae live in symbiosis with intracellular chemosynthetic bacteria. These symbionts are transmitted maternally (vertically) between host generations and should therefore show a pattern of genetic variation paralleling that of the cotransmitted host mitochondrion. However, instances of lateral (nonvertical) symbiont acquisition could still occur, thereby decoupling symbiont and mitochondrial phylogenies. Here, we provide the first evidence against strict maternal cotransmission of symbiont and mitochondrial genomes in vesicomyids. Analysis of Vesicomya sp. mt-II clams from hydrothermal vents on the Juan de Fuca Ridge (northeastern Pacific) revealed a symbiont phylotype (designated symB(VII)) highly divergent from previously described symbionts of the same host lineage. SymB(VII)-hosting clams occurred at low frequency (0.02) relative to individuals hosting the dominant symbiont phylotype. Phylogenetic analysis of 16S rRNA genes from a wide range of symbionts and free-living bacteria clustered symB(VII) within the monophyletic clade of vesicomyid symbionts. Further analysis of 3 symbiont loci (23S, dnaK, and soxA) across 11 vesicomyid taxa unambiguously placed symB(VII) as sister to the symbiont of a distantly related host lineage, Vesicomya sp. from the Mid-Atlantic Ridge (98.9% median nucleotide identity across protein-coding loci). Using likelihood and Bayesian model discrimination methods, we rejected the strict maternal cotransmission hypothesis by showing a significant decoupling of symbiont and host mitochondrial (COI and mt16S genes) phylogenies. Indeed, decoupling occurred even when symB(VII) was excluded from phylogenetic reconstructions, suggesting a history of host switching in this group. Together, the data indicate a history of lateral symbiont transfer in vesicomyids, with symB(VII) being the most conspicuous example. Interpreted alongside previous studies of the vesicomyid symbiosis, these results suggest a mixed mode of symbiont transmission characterized by predominantly vertical transmission punctuated with instances of lateral symbiont acquisition. Lateral acquisition may facilitate the exchange of genetic material (recombination) among divergent symbiont lineages, rendering the evolutionary history of vesicomyid symbiont genomes much more complex than previously thought.

Migration, isolation, and speciation of hydrothermal vent limpets (Gastropoda; Lepetodrilidae) across the Blanco Transform Fault.

The Sovanco Fracture Zone and Blanco Transform Fault separate the Explorer, Juan de Fuca, and Gorda ridge systems of the northeastern Pacific Ocean. To test whether such offsets in the ridge axis create barriers to along-axis dispersal of the endemic hydrothermal vent animals, we examined the genetic structure of limpet populations previously identified as Lepetodrilus fucensis McLean, 1988 (Gastropoda, Lepetodrilidae). Mitochondrial DNA sequences and patterns of allozyme variation revealed no evidence that the 150-km-long Sovanco Fracture Zone impeded gene flow between the Explorer and Juan de Fuca populations. In contrast, the 450-km-long Blanco Transform Fault separates the limpets into highly divergent northern and southern lineages that we recognize as distinct species. We describe southern populations from the Gorda Ridge (Seacliff) and Escanaba Trough as Lepetodrilus gordensis new species and refer northern populations from the Explorer and Juan de Fuca ridge systems to L. fucensis sensu stricto. The species are similar morphologically, but L. gordensis lacks a sensory neck papilla and has a more tightly coiled teleconch. To assess the degree of isolation between these closely related species, we used the Isolation with Migration method to estimate the time of population splitting, effective sizes of the ancestral and derived populations, and rates of migration across the Blanco Transform Fault.

Ocean microbes. Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages.

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Experimental incubations elicit profound changes in community transcription in OMZ bacterioplankton.

Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for "challenging" microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4-13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations.

Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium.

Proteorhodopsin (PR) is a photoprotein that functions as a light-driven proton pump in diverse marine Bacteria and Archaea. Recent studies have suggested that PR may enhance both growth rate and yield in some flavobacteria when grown under nutrient-limiting conditions in the light. The direct involvement of PR, and the metabolic details enabling light-stimulated growth, however, remain uncertain. Here, we surveyed transcriptional and growth responses of a PR-containing marine flavobacterium during carbon-limited growth in the light and the dark. As previously reported (Gómez-Consarnau et al., 2007), Dokdonia strain MED134 exhibited light-enhanced growth rates and cell yields under low carbon growth conditions. Inhibition of retinal biosynthesis abolished the light-stimulated growth response, supporting a direct role for retinal-bound PR in light-enhanced growth. Among protein-coding transcripts, both PR and retinal biosynthetic enzymes showed significant upregulation in the light. Other light-associated proteins, including bacterial cryptochrome and DNA photolyase, were also expressed at significantly higher levels in the light. Membrane transporters for Na(+)/phosphate and Na(+)/alanine symporters, and the Na(+)-translocating NADH-quinone oxidoreductase (NQR) linked electron transport chain, were also significantly upregulated in the light. Culture experiments using a specific inhibitor of Na(+)-translocating NQR indicated that sodium pumping via NQR is a critical metabolic process in the light-stimulated growth of MED134. In total, the results suggested the importance of both the PR-enabled, light-driven proton gradient, as well as the generation of a Na(+) ion gradient, as essential components for light-enhanced growth in these flavobacteria.

Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton.

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.