Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.
Proceedings of the National Academy of Sciences. 10.1073/pnas.1501615112 Abstract
Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops.
Using community metabolomics as a new approach to discriminate marine microbial particulate organic matter in the western English Channel.
Progress in Oceanography. 10.1016/j.pocean.2015.04.022 Abstract
Metabolomics provides an unbiased assessment of a wide range of metabolites and is an emerging ‘omics technique in the marine sciences. We use ‘non-targeted’ community metabolomics to determine patterns in metabolite profiles associated with particulate organic matter (POM) at four locations from two long-term monitoring stations (L4 and E1) in the western English Channel. The polar metabolite fractions were measured using ultra-high performance liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS), and the lipid fractions by direct infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS); these were then analysed to statistically compare the metabolite distributions. Results show significantly different profiles of metabolites across the four locations with the largest differences for both the polar and lipid fractions found between the two stations relative to the smaller differences associated with depth. We putatively annotate the most discriminant metabolites revealing a range of amino-acid derivatives, diacylglyceryltrimethylhomoserine (DGTS) lipids, oxidised fatty acids (oxylipins), glycosylated compounds, oligohexoses, phospholipids, triacylglycerides (TAGs) and oxidised TAGs. The majority of the polar metabolites were most abundant in the surface waters at L4 and least abundant in the deep waters at E1 (E1-70m). In contrast, the oxidised TAGs were more abundant at E1 and most abundant at E1-70m. The differentiated metabolites are discussed in relation to the health of the phytoplankton as indicated by nutrients, carbon and chlorophyll, and to the dominance (determined from metatranscript data) of the picoeukaryote Ostreococcus. Our results show proof of concept for community metabolomics in discriminating and characterising polar and lipid metabolite patterns associated with marine POM.
Designer diatom episomes delivered by bacterial conjugation.
Nat Commun. 6: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 10.1038/ncomms7925 Abstract
Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.
A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake.
Current Biology. 25:364-371. 10.1016/j.cub.2014.12.004 Abstract
Summary Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean . Iron bioavailability has drastically decreased in the contemporary ocean , most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms , indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3–5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.
Vitamin B1 ecophysiology of marine picoeukaryotic algae: Strain-specific differences and a new role for bacteria in vitamin cycling.
Limnology and Oceanography. 60:215-228. 10.1002/lno.10009 Abstract
We confirmed multiple picoeukaryotic algae, Ostreococcus, Micromonas, and Pelagomonas spp., as thiamine (vitamin B1) auxotrophs in laboratory experiments with axenic cultures. Examined strains have half saturation growth constants (K-s) for B1 between 1.26 and 6.22 pmol B1 L-1, which is higher than reported seawater concentrations. Minimum B1 cell quotas for Ostreococcus and Micromonas spp. are high (2.20 x 10(-8)-4.46 x 10(-8) pmol B1 cell(-1)) relative to other B1 auxotrophic phytoplankton, potentially making them B1 rich prey for zooplankton and significant B1 reservoirs in oligotrophic marine habitats. Ostreococcus and Micromonas genomes are nonuniformly missing portions of the B1 biosynthesis pathway. Given their gene repertoires, Ostreococcus lucimarinus CCE9901 and Ostreococcus tauri OTH95 are expected to salvage B1 from externally provided 4-methyl-5-thiazoleethanol (HET) and 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). However, in culture, neither could use HET plus HMP instead of B1, highlighting current limitations of genome-based prediction of B1 salvaging by picoeukaryotic algae. HMP and phosphorylated B1 use varied amongst tested strains and notably all Prasinophytes tested could not use HMP. B1-limited O. lucimarinus CCE9901 could not grow on added thiamine diphosphate (TDP), a phosophorylated B1 form. However, in co-culture with Pseudoalteromonas sp. TW7, a bacterium known to exhibit phosphatase activity, O. lucimarinus CCE9901 exhibited increased growth following TDP additions. This demonstrates that bacteria influence vitamin B1 availability beyond de novo synthesis and consumption; they can also serve as conduits that chemically alter, but not completely degrade or retain B1 analogs (e.g., TDP), and make them accessible to a broader range of microbes.
Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities.
ISME J. 9:1076-1092.: International Society for Microbial Ecology 10.1038/ismej.2014.198 Abstract
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-[mu]m 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.
Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.
Plant Biotechnology Journal. 13:460-470. 10.1111/pbi.12254 Abstract
Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.