Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates.

Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (<0.01-1%) of vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time.

Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 µm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

Using dispersants after oil spills: impacts on the composition and activity of microbial communities.

Dispersants are globally and routinely applied as an emergency response to oil spills in marine ecosystems with the goal of chemically enhancing the dissolution of oil into water, which is assumed to stimulate microbially mediated oil biodegradation. However, little is known about how dispersants affect the composition of microbial communities or their biodegradation activities. The published findings are controversial, probably owing to variations in laboratory methods, the selected model organisms and the chemistry of different dispersant-oil mixtures. Here, we argue that an in-depth assessment of the impacts of dispersants on microorganisms is needed to evaluate the planning and use of dispersants during future responses to oil spills.

Microspatial gene expression patterns in the Amazon River Plume.

We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean's largest river plume using, to our knowledge, the first fully quantitative inventory of genes and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼ 1.0 × 10(13) genes and 4.7 × 10(11) transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts ⋅ gene(-1)) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.

The Amazon continuum dataset: quantitative metagenomic and metatranscriptomic inventories of the Amazon River plume, June 2010.

BACKGROUND: The Amazon River is by far the world's largest in terms of volume and area, generating a fluvial export that accounts for about a fifth of riverine input into the world's oceans. Marine microbial communities of the Western Tropical North Atlantic Ocean are strongly affected by the terrestrial materials carried by the Amazon plume, including dissolved (DOC) and particulate organic carbon (POC) and inorganic nutrients, with impacts on primary productivity and carbon sequestration. RESULTS: We inventoried genes and transcripts at six stations in the Amazon River plume during June 2010. At each station, internal standard-spiked metagenomes, non-selective metatranscriptomes, and poly(A)-selective metatranscriptomes were obtained in duplicate for two discrete size fractions (0.2 to 2.0 µm and 2.0 to 156 µm) using 150 × 150 paired-end Illumina sequencing. Following quality control, the dataset contained 360 million reads of approximately 200 bp average size from Bacteria, Archaea, Eukarya, and viruses. Bacterial metagenomes and metatranscriptomes were dominated by Synechococcus, Prochlorococcus, SAR11, SAR116, and SAR86, with high contributions from SAR324 and Verrucomicrobia at some stations. Diatoms, green picophytoplankton, dinoflagellates, haptophytes, and copepods dominated the eukaryotic genes and transcripts. Gene expression ratios differed by station, size fraction, and microbial group, with transcription levels varying over three orders of magnitude across taxa and environments. CONCLUSIONS: This first comprehensive inventory of microbial genes and transcripts, benchmarked with internal standards for full quantitation, is generating novel insights into biogeochemical processes of the Amazon plume and improving prediction of climate change impacts on the marine biosphere.

Comparative metagenomics: natural populations of induced prophages demonstrate highly unique, lower diversity viral sequences.

To understand the similarities and differences between a free living viral population and its co-occurring temperate population, metagenomes of each type were prepared from the same seawater sample from Tampa Bay, FL. Libraries were prepared from extracted DNA of the ambient viruses and induced prophages from the co-occurring, viral-reduced microbial assemblage. Duplicate libraries were also prepared using the same DNA amplified by multiple displacement amplification. A non-viral-reduced, induced, amplified viral dataset from the same site in 2005 was reanalysed for temporal comparison. The induced viral metagenome was higher in identifiable virus sequences and differed from the other three datasets based on principal component, rarefaction, trinucleotide composition and contig spectrum analyses. This study indicated that induced prophages are unique and have lower overall community diversity than ambient viral populations from the same site. Both of the amplified contemporary metagenomes were enriched in single-stranded DNA (ssDNA) viral sequences. Six and 16 complete, circular ssDNA viral genomes were assembled from the amplified induced and ambient libraries, respectively, mostly similar to circoviruses. The amplified ambient metagenome contained genomes similar to an RNA-DNA hybrid virus recently identified in a hot spring and to an ssDNA virus infecting the diatom Chaetoceros.

Toxicity and mutagenicity of Gulf of Mexico waters during and after the deepwater horizon oil spill.

The Deepwater Horizon oil spill is unparalleled among environmental hydrocarbon releases, because of the tremendous volume of oil, the additional contamination by dispersant, and the oceanic depth at which this release occurred. Here, we present data on general toxicity and mutagenicity of upper water column waters and, to a lesser degree, sediment porewater of the Northeastern Gulf of Mexico (NEGOM) and west Florida shelf (WFS) at the time of the Deepwater Horizon oil spill in 2010 and thereafter. During a research cruise in August 2010, analysis of water collected in the NEGOM indicated that samples of 3 of 14 (21%) stations were toxic to bacteria based on the Microtox assay, 4 of 13 (34%) were toxic to phytoplankton via the QwikLite assay, and 6 of 14 (43%) showed DNA damaging activity using the λ-Microscreen Prophage induction assay. The Microtox and Microscreen assays indicated that the degree of toxicity was correlated to total petroleum hydrocarbon concentration. Long-term monitoring of stations on the NEGOM and the WFS was undertaken by 8 and 6 cruises to these areas, respectively. Microtox toxicity was nearly totally absent by December 2010 in the Northeastern Gulf of Mexico (3 of 8 cruises with one positive station). In contrast, QwikLite toxicity assay yielded positives at each cruise, often at multiple stations or depths, indicating the greater sensitivity of the QwikLite assay to environmental factors. The Microscreen mutagenicity assays indicated that certain water column samples overlying the WFS were mutagenic at least 1.5 years after capping the Macondo well. Similarly, sediment porewater samples taken from 1000, 1200, and 1400 m from the slope off the WFS in June 2011 were also highly genotoxic. Our observations are consistent with a portion of the dispersed oil from the Macondo well area advecting to the southeast and upwelling onto the WFS, although other explanations exist. Organisms in contact with these waters might experience DNA damage that could lead to mutation and heritable alterations to the community pangenome. Such mutagenic interactions might not become apparent in higher organisms for years.

Environmental factors influencing gene transfer agent (GTA) mediated transduction in the subtropical ocean.

Microbial genomic sequence analyses have indicated widespread horizontal gene transfer (HGT). However, an adequate mechanism accounting for the ubiquity of HGT has been lacking. Recently, high frequencies of interspecific gene transfer have been documented, catalyzed by Gene Transfer Agents (GTAs) of marine α-Proteobacteria. It has been proposed that the presence of bacterial genes in highly purified viral metagenomes may be due to GTAs. However, factors influencing GTA-mediated gene transfer in the environment have not yet been determined. Several genomically sequenced strains containing complete GTA sequences similar to Rhodobacter capsulatus (RcGTA, type strain) were screened to ascertain if they produced putative GTAs, and at what abundance. Five of nine marine strains screened to date spontaneously produced virus-like particles (VLP's) in stationary phase. Three of these strains have demonstrated gene transfer activity, two of which were documented by this lab. These two strains Roseovarius nubinhibens ISM and Nitratireductor 44B9s, were utilized to produce GTAs designated RnGTA and NrGTA and gene transfer activity was verified in culture. Cell-free preparations of purified RnGTA and NrGTA particles from marked donor strains were incubated with natural microbial assemblages to determine the level of GTA-mediated gene transfer. In conjunction, several ambient environmental parameters were measured including lysogeny indicated by prophage induction. GTA production in culture systems indicated that approximately half of the strains produced GTA-like particles and maximal GTA counts ranged from 10-30% of host abundance. Modeling of GTA-mediated gene transfer frequencies in natural samples, along with other measured environmental variables, indicated a strong relationship between GTA mediated gene transfer and the combined factors of salinity, multiplicity of infection (MOI) and ambient bacterial abundance. These results indicate that GTA-mediated HGT in the marine environment with the strains examined is favored during times of elevated bacterial and GTA abundance as well as in areas of higher salinity.

High frequency of horizontal gene transfer in the oceans.

Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

Functional prokaryotic RubisCO from an oceanic metagenomic library.

Culture-independent studies have indicated that there is significant diversity in the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzymes used by marine, freshwater, and terrestrial autotrophic bacteria. Surprisingly, little is known about the catalytic properties of many environmentally significant RubisCO enzymes. Because one of the goals of RubisCO research is to somehow modify or select for RubisCO molecules with improved kinetic properties, a facile means to isolate functional and novel RubisCO molecules directly from the environment was developed. In this report, we describe the first example of functional RubisCO proteins obtained from genes cloned and characterized from metagenomic libraries derived from DNA isolated from environmental samples. Two form IA marine RubisCO genes were cloned, and each gene supported both photoheterotrophic and photoautotrophic growth of a RubisCO deletion strain of Rhodobacter capsulatus, strain SBI/II(-), indicating that catalytically active recombinant RubisCO was synthesized. The catalytic properties of the metagenomic RubisCO molecules were further characterized. These experiments demonstrated the feasibility of studying the functional diversity and enzymatic properties of RubisCO enzymes without first cultivating the host organisms. Further, this "proof of concept" experiment opens the way for development of a simple functional screen to examine the properties of diverse RubisCO genes isolated from any environment, and subsequent further bioselection may be possible if the growth conditions of complemented R. capsulatus strain SBI/II(-) are varied.

Quantification of human polyomaviruses JC Virus and BK Virus by TaqMan quantitative PCR and comparison to other water quality indicators in water and fecal samples.

In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify > or =10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35 degrees C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.

Metagenomic analysis of lysogeny in Tampa Bay: implications for prophage gene expression.

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e < or =0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9 x 10(7), and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L(-1) respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L(-1). Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L(-1). In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.

Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas?

Bacteriophages are realized to be numerous and important components of oceanic food webs principally because of their lytic capabilities. The subtle changes that temperate phages impart to their hosts in the oceans are far less understood. Occurrences of lysogeny in the oceans correlate well with conditions unfavorable for rapid host growth. In coliphage lambda, phage encoded repressors have been shown to modulate host metabolic gene expression and phenotype, resulting in economizing host energy expenditure. Comparison of lysogenized marine bacteria to the uninfected hosts indicated that prophage acquisition is correlated with host metabolic gene suppression. Screening 113 marine bacterial genomes for prophages yielded 64 prophage-like elements, 21 of which strongly resembled gene transfer agents (GTAs). The remaining 39 putative prophages had a relatively high incidence of transcriptional regulatory and repressor-like proteins (approximately 2/40 kb prophage sequence) compared to lytic marine phages (approximately 0.25/40 kb phage sequence). Here, it has been hypothesized that marine prophages directly contribute to host survival in unfavorable environments by suppression of unneeded metabolic activities. It has been further suggested that such metabolic downshifts are the result of phage-encoded repressors and transcriptional regulators acting directly on host genes. Finally, the widespread occurrence of GTAs may be an efficient mechanism for horizontal gene transfer in the oceans.

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome.

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

Comparison of lysogeny (prophage induction) in heterotrophic bacterial and Synechococcus populations in the Gulf of Mexico and Mississippi River plume.

Lysogeny has been documented as a fundamental process occurring in natural marine communities of heterotrophic and autotrophic bacteria. Prophage induction has been observed to be prevalent during conditions of low host abundance, but factors controlling the process are poorly understood. A research cruise was undertaken to the Gulf of Mexico during July 2005 to explore environmental factors associated with lysogeny. Ambient physical and microbial parameters were measured and prophage induction experiments were performed in contrasting oligotrophic Gulf and eutrophic Mississippi plume areas. Three of 11 prophage induction experiments in heterotrophic bacteria (27%) demonstrated significant induction in response to Mitomycin C. In contrast, there was significant Synechococcus cyanophage induction in seven of nine experiments (77.8%). A strong negative correlation was observed between lysogeny and log-transformed activity measurements for both heterotrophic and autotrophic populations (r=-0.876, P=0.002 and r=-0.815, P=0.025, respectively), indicating that bacterioplankton with low host growth favor lysogeny. Multivariate statistical analyses indicated that ambient level of viral abundance and productivity were inversely related to heterotrophic prophage induction and both factors combined were most predictive of lysogeny (rho=0.899, P=0.001). For Synechococcus, low ambient cyanophage abundance was most predictive of lysogeny (rho=0.862, P=0.005). Abundance and productivity of heterotrophic bacteria was strongly inversely correlated with salinity, while Synechococcus was not. This indicated that heterotrophic bacterial populations were well adapted to the river plume environments, thus providing a possible explanation for differences in prevalence of lysogeny observed between the two populations.

Phytoplankton carbon fixation gene (RuBisCO) transcripts and air-sea CO(2) flux in the Mississippi River plume.

River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in significant CO(2) drawdown. To determine the relationship between expression of the major gene in carbon fixation (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) and CO(2) dynamics, we evaluated rbcL mRNA abundance using novel quantitative PCR assays, phytoplankton cell analyses, photophysiological parameters, and pCO(2) in and around the Mississippi River plume (MRP) in the Gulf of Mexico. Lower salinity (30-32) stations were dominated by rbcL mRNA concentrations from heterokonts, such as diatoms and pelagophytes, which were at least an order of magnitude greater than haptophytes, alpha-Synechococcus or high-light Prochlorococcus. However, rbcL transcript abundances were similar among these groups at oligotrophic stations (salinity 34-36). Diatom cell counts and heterokont rbcL RNA showed a strong negative correlation to seawater pCO(2). While Prochlorococcus cells did not exhibit a large difference between low and high pCO(2) water, Prochlorococcus rbcL RNA concentrations had a strong positive correlation to pCO(2), suggesting a very low level of RuBisCO RNA transcription among Prochlorococcus in the plume waters, possibly due to their relatively poor carbon concentrating mechanisms (CCMs). These results provide molecular evidence that diatom/pelagophyte productivity is largely responsible for the large CO(2) drawdown occurring in the MRP, based on the co-occurrence of elevated RuBisCO gene transcript concentrations from this group and reduced seawater pCO(2) levels. This may partly be due to efficient CCMs that enable heterokont eukaryotes such as diatoms to continue fixing CO(2) in the face of strong CO(2) drawdown. Our work represents the first attempt to relate in situ microbial gene expression to contemporaneous CO(2) flux measurements in the ocean.

Phytoplankton-group specific quantitative polymerase chain reaction assays for RuBisCO mRNA transcripts in seawater.

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.