Nathan G. Walworth

Irreversibly increased nitrogen fixation in Trichodesmium experimentally adapted to elevated carbon dioxide

Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.

Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean

Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean.

Trichodesmium genome maintains abundant, widespread noncoding DNA in situ, despite oligotrophic lifestyle

Significance The free-living cyanobacterium Trichodesmium is a major source of new nitrogen and fixed carbon to the tropical and subtropical oceans, but despite its importance, we know little about the molecular mechanisms it uses to succeed in its oligotrophic habitat. Here we show that its gene-sparse genome is littered with large, conserved, expressed intergenic spaces, which is atypical for most known free-living prokaryotes. Paradoxically, although its genome is enriched in predicted transposases and repeat sequences, it exhibits conserved intragenus synteny and similar intergenic architecture relative to its sympatric, gene-dense relatives Prochlorococcus and Synechococcus. This observation demonstrates a successful alternative to the genomic streamlining strategy observed in other free-living oligotrophs such as Prochlorococcus or Pelagibacter. Understanding the evolution of the free-living, cyanobacterial, diazotroph Trichodesmium is of great importance because of its critical role in oceanic biogeochemistry and primary production. Unlike the other >150 available genomes of free-living cyanobacteria, only 63.8% of the Trichodesmium erythraeum (strain IMS101) genome is predicted to encode protein, which is 20–25% less than the average for other cyanobacteria and nonpathogenic, free-living bacteria. We use distinctive isolates and metagenomic data to show that low coding density observed in IMS101 is a common feature of the Trichodesmium genus, both in culture and in situ. Transcriptome analysis indicates that 86% of the noncoding space is expressed, although the function of these transcripts is unclear. The density of noncoding, possible regulatory elements predicted in Trichodesmium, when normalized per intergenic kilobase, was comparable and twofold higher than that found in the gene-dense genomes of the sympatric cyanobacterial genera Synechococcus and Prochlorococcus, respectively. Conserved Trichodesmium noncoding RNA secondary structures were predicted between most culture and metagenomic sequences, lending support to the structural conservation. Conservation of these intergenic regions in spatiotemporally separated Trichodesmium populations suggests possible genus-wide selection for their maintenance. These large intergenic spacers may have developed during intervals of strong genetic drift caused by periodic blooms of a subset of genotypes, which may have reduced effective population size. Our data suggest that transposition of selfish DNA, low effective population size, and high-fidelity replication allowed the unusual “inflation” of noncoding sequence observed in Trichodesmium despite its oligotrophic lifestyle.

The Trichodesmium consortium: conserved heterotrophic co-occurrence and genomic signatures of potential interactions

The nitrogen (N)-fixing cyanobacterium Trichodesmium is globally distributed in warm, oligotrophic oceans, where it contributes a substantial proportion of new N and fuels primary production. These photoautotrophs form macroscopic colonies that serve as relatively nutrient-rich substrates that are colonized by many other organisms. The nature of these associations may modulate ocean N and carbon (C) cycling, and can offer insights into marine co-evolutionary mechanisms. Here we integrate multiple omics-based and experimental approaches to investigate Trichodesmium-associated bacterial consortia in both laboratory cultures and natural environmental samples. These efforts have identified the conserved presence of a species of Gammaproteobacteria (Alteromonas macleodii), and enabled the assembly of a near-complete, representative genome. Interorganismal comparative genomics between A. macleodii and Trichodesmium reveal potential interactions that may contribute to the maintenance of this association involving iron and phosphorus acquisition, vitamin B12 exchange, small C compound catabolism, and detoxification of reactive oxygen species. These results identify what may be a keystone organism within Trichodesmium consortia and support the idea that functional selection has a major role in structuring associated microbial communities. These interactions, along with likely many others, may facilitate Trichodesmium’s unique open-ocean lifestyle, and could have broad implications for oligotrophic ecosystems and elemental cycling.

Molecular and physiological evidence of genetic assimilation to high CO2 in the marine nitrogen fixer Trichodesmium

Significance The free-living cyanobacterium Trichodesmium is an important nitrogen-fixer in the global oceans, yet virtually nothing is known about its molecular evolution to increased CO2. Here we show that Trichodesmium can fix a plastic, short-term response upon long-term adaptation, potentially through genetic assimilation. We provide transcriptional evidence for molecular mechanisms that parallel the fixation of the plastic phenotype, thereby demonstrating an important evolutionary capability in Trichodesmium CO2 adaptation. Transcriptional shifts involve transposition and other regulatory mechanisms (sigma factors) that control a variety of metabolic pathways, suggesting alterations in upstream regulation to be important under genetic assimilation. Together, these data highlight potential biochemical evidence of genetic assimilation in a keystone marine N2-fixer, with broad implications for microbial evolution and biogeochemistry. Most investigations of biogeochemically important microbes have focused on plastic (short-term) phenotypic responses in the absence of genetic change, whereas few have investigated adaptive (long-term) responses. However, no studies to date have investigated the molecular progression underlying the transition from plasticity to adaptation under elevated CO2 for a marine nitrogen-fixer. To address this gap, we cultured the globally important cyanobacterium Trichodesmium at both low and high CO2 for 4.5 y, followed by reciprocal transplantation experiments to test for adaptation. Intriguingly, fitness actually increased in all high-CO2 adapted cell lines in the ancestral environment upon reciprocal transplantation. By leveraging coordinated phenotypic and transcriptomic profiles, we identified expression changes and pathway enrichments that rapidly responded to elevated CO2 and were maintained upon adaptation, providing strong evidence for genetic assimilation. These candidate genes and pathways included those involved in photosystems, transcriptional regulation, cell signaling, carbon/nitrogen storage, and energy metabolism. Conversely, significant changes in specific sigma factor expression were only observed upon adaptation. These data reveal genetic assimilation as a potentially adaptive response of Trichodesmium and importantly elucidate underlying metabolic pathways paralleling the fixation of the plastic phenotype upon adaptation, thereby contributing to the few available data demonstrating genetic assimilation in microbial photoautotrophs. These molecular insights are thus critical for identifying pathways under selection as drivers in plasticity and adaptation.

Microbial Communities on Seafloor Basalts at Dorado Outcrop Reflect Level of Alteration and Highlight Global Lithic Clades

Areas of exposed basalt along mid-ocean ridges and at seafloor outcrops serve as conduits of fluid flux into and out of a subsurface ocean, and microbe–mineral interactions can influence alteration reactions at the rock–water interface. Located on the eastern flank of the East Pacific Rise, Dorado Outcrop is a site of low-temperature (<20°C) hydrothermal venting and represents a new end-member in the current survey of seafloor basalt biomes. Consistent with prior studies, a survey of 16S rRNA gene sequence diversity using universal primers targeting the V4 hypervariable region revealed much greater richness and diversity on the seafloor rocks than in surrounding seawater. Overall, Gamma-, Alpha-, and Deltaproteobacteria, and Thaumarchaeota dominated the sequenced communities, together making up over half of the observed diversity, though bacterial sequences were more abundant than archaeal in all samples. The most abundant bacterial reads were closely related to the obligate chemolithoautotrophic, sulfur-oxidizing Thioprofundum lithotrophicum, suggesting carbon and sulfur cycling as dominant metabolic pathways in this system. Representatives of Thaumarchaeota were detected in relatively high abundance on the basalts in comparison to bottom water, possibly indicating ammonia oxidation. In comparison to other sequence datasets from globally distributed seafloor basalts, this study reveals many overlapping and cosmopolitan phylogenetic groups and also suggests that substrate age correlates with community structure.

Comment on “The complex effects of ocean acidification on the prominent N2-fixing cyanobacterium Trichodesmium”

Hong et al. (Reports, 5 May 2017, p. 527) suggested that previous studies of the biogeochemically significant marine cyanobacterium Trichodesmium showing increased growth and nitrogen fixation at projected future high CO2 levels suffered from ammonia or copper toxicity. They reported that these rates instead decrease at high CO2 when contamination is alleviated. We present and discuss results of multiple published studies refuting this toxicity hypothesis.

Interactive effects of temperature, CO2 and nitrogen source on a coastal California diatom assemblage

ENVIRONMENTAL GENOMICS, J. CRAIG VENTER INSTITUTE, LA JOLLA, CA , USA, INTEGRATIVE OCEANOGRAPHY DIVISION, SCRIPPS INSTITUTION OF OCEANOGRAPHY, UC SAN DIEGO, LA JOLLA, CA , USA, STATE KEY LABORATORY OF MARINE ENVIRONMENTAL SCIENCE, XIAMEN UNIVERSITY, XIAMEN, FUJIAN , PR CHINA AND COLLEGE OF MARINE AND ENVIRONMENTAL SCIENCES, TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY, TIANJIN ,

Transcriptional Activities of the Microbial Consortium Living with the Marine Nitrogen-Fixing Cyanobacterium Trichodesmium Reveal Potential Roles in Community-Level Nitrogen Cycling

ABSTRACT Trichodesmium is a globally distributed cyanobacterium whose nitrogen-fixing capability fuels primary production in warm oligotrophic oceans. Like many photoautotrophs, Trichodesmium serves as a host to various other microorganisms, yet little is known about how this associated community modulates fluxes of environmentally relevant chemical species into and out of the supraorganismal structure. Here, we utilized metatranscriptomics to examine gene expression activities of microbial communities associated with Trichodesmium erythraeum (strain IMS101) using laboratory-maintained enrichment cultures that have previously been shown to harbor microbial communities similar to those of natural populations. In enrichments maintained under two distinct CO2 concentrations for ∼8 years, the community transcriptional profiles were found to be specific to the treatment, demonstrating a restructuring of overall gene expression had occurred. Some of this restructuring involved significant increases in community respiration-related transcripts under elevated CO2, potentially facilitating the corresponding measured increases in host nitrogen fixation rates. Particularly of note, in both treatments, community transcripts involved in the reduction of nitrate, nitrite, and nitrous oxide were detected, suggesting the associated organisms may play a role in colony-level nitrogen cycling. Lastly, a taxon-specific analysis revealed distinct ecological niches of consistently cooccurring major taxa that may enable, or even encourage, the stable cohabitation of a diverse community within Trichodesmium consortia. IMPORTANCE Trichodesmium is a genus of globally distributed, nitrogen-fixing marine cyanobacteria. As a source of new nitrogen in otherwise nitrogen-deficient systems, these organisms help fuel carbon fixation carried out by other more abundant photoautotrophs and thereby have significant roles in global nitrogen and carbon cycling. Members of the Trichodesmium genus tend to form large macroscopic colonies that appear to perpetually host an association of diverse interacting microbes distinct from the surrounding seawater, potentially making the entire assemblage a unique miniature ecosystem. Since its first successful cultivation in the early 1990s, there have been questions about the potential interdependencies between Trichodesmium and its associated microbial community and whether the hosts seemingly enigmatic nitrogen fixation schema somehow involved or benefited from its epibionts. Here, we revisit these old questions with new technology and investigate gene expression activities of microbial communities living in association with Trichodesmium.