Erin M. Bertrand

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

The Transcriptome and Proteome of the Diatom Thalassiosira pseudonana Reveal a Diverse Phosphorus Stress Response

Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem function in the ocean. Diatoms are an abundant class of marine phytoplankton that are responsible for significant amounts of primary production. With the control they exert on the oceanic carbon cycle, there have been a number of studies focused on how diatoms respond to limiting macro and micronutrients such as iron and nitrogen. However, diatom physiological responses to P deficiency are poorly understood. Here, we couple deep sequencing of transcript tags and quantitative proteomics to analyze the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. A total of 318 transcripts were differentially regulated with a false discovery rate of <0.05, and a total of 136 proteins were differentially abundant (p<0.05). Significant changes in the abundance of transcripts and proteins were observed and coordinated for multiple biochemical pathways, including glycolysis and translation. Patterns in transcript and protein abundance were also linked to physiological changes in cellular P distributions, and enzyme activities. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P through increased production of metalloenzymes, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to respond to variations in environmental P availability.

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii

The marine nitrogen fixing microorganisms (diazotrophs) are a major source of nitrogen to open ocean ecosystems and are predicted to be limited by iron in most marine environments. Here we use global and targeted proteomic analyses on a key unicellular marine diazotroph Crocosphaera watsonii to reveal large scale diel changes in its proteome, including substantial variations in concentrations of iron metalloproteins involved in nitrogen fixation and photosynthesis, as well as nocturnal flavodoxin production. The daily synthesis and degradation of enzymes in coordination with their utilization results in a lowered cellular metalloenzyme inventory that requires ∼40% less iron than if these enzymes were maintained throughout the diel cycle. This strategy is energetically expensive, but appears to serve as an important adaptation for confronting the iron scarcity of the open oceans. A global numerical model of ocean circulation, biogeochemistry and ecosystems suggests that Crocosphaera’s ability to reduce its iron-metalloenzyme inventory provides two advantages: It allows Crocosphaera to inhabit regions lower in iron and allows the same iron supply to support higher Crocosphaera biomass and nitrogen fixation than if they did not have this reduced iron requirement.

Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein

Diatoms are responsible for ∼40% of marine primary production and are key players in global carbon cycling. There is mounting evidence that diatom growth is influenced by cobalamin (vitamin B12) availability. This cobalt-containing micronutrient is only produced by some bacteria and archaea but is required by many diatoms and other eukaryotic phytoplankton. Despite its potential importance, little is known about mechanisms of cobalamin acquisition in diatoms or the impact of cobalamin scarcity on diatom molecular physiology. Proteomic profiling and RNA-sequencing transcriptomic analysis of the cultured diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana revealed three distinct strategies used by diatoms to cope with low cobalamin: increased cobalamin acquisition machinery, decreased cobalamin demand, and management of reduced methionine synthase activity through changes in folate and S-adenosyl methionine metabolism. One previously uncharacterized protein, cobalamin acquisition protein 1 (CBA1), was up to 160-fold more abundant under low cobalamin availability in both diatoms. Autologous overexpression of CBA1 revealed association with the outside of the cell and likely endoplasmic reticulum localization. Cobalamin uptake rates were elevated in strains overexpressing CBA1, directly linking this protein to cobalamin acquisition. CBA1 is unlike characterized cobalamin acquisition proteins and is the only currently identified algal protein known to be implicated in cobalamin uptake. The abundance and widespread distribution of transcripts encoding CBA1 in environmental samples suggests that cobalamin is an important nutritional factor for phytoplankton. Future study of CBA1 and other molecular signatures of cobalamin scarcity identified here will yield insight into the evolution of cobalamin utilization and facilitate monitoring of cobalamin starvation in oceanic diatom communities.

Proteome Changes Driven by Phosphorus Deficiency and Recovery in the Brown Tide-Forming Alga Aureococcus anophagefferens

Shotgun mass spectrometry was used to detect proteins in the harmful alga, Aureococcus anophagefferens, and monitor their relative abundance across nutrient replete (control), phosphate-deficient (−P) and −P refed with phosphate (P-refed) conditions. Spectral counting techniques identified differentially abundant proteins and demonstrated that under phosphate deficiency, A. anophagefferens increases proteins involved in both inorganic and organic phosphorus (P) scavenging, including a phosphate transporter, 5′-nucleotidase, and alkaline phosphatase. Additionally, an increase in abundance of a sulfolipid biosynthesis protein was detected in −P and P-refed conditions. Analysis of the polar membrane lipids showed that cellular concentrations of the sulfolipid sulphoquinovosyldiacylglycerol (SQDG) were nearly two-fold greater in the −P condition versus the control condition, while cellular phospholipids were approximately 8-fold less. Transcript and protein abundances were more tightly coupled for gene products involved in P metabolism compared to those involved in a range of other metabolic functions. Comparison of protein abundances between the −P and P-refed conditions identified differences in the timing of protein degradation and turnover. This suggests that culture studies examining nutrient starvation responses will be valuable in interpreting protein abundance patterns for cellular nutritional status and history in metaproteomic datasets.

Influence of vitamin B auxotrophy on nitrogen metabolism in eukaryotic phytoplankton

While nitrogen availability is known to limit primary production in large parts of the ocean, vitamin starvation amongst eukaryotic phytoplankton is becoming increasingly recognized as an oceanographically relevant phenomenon. Cobalamin (B12) and thiamine (B1) auxotrophy are widespread throughout eukaryotic phytoplankton, with over 50% of cultured isolates requiring B12 and 20% requiring B1. The frequency of vitamin auxotrophy in harmful algal bloom species is even higher. Instances of colimitation between nitrogen and B vitamins have been observed in marine environments, and interactions between these nutrients have been shown to impact phytoplankton species composition. This review surveys available data, including relevant gene expression patterns, to evaluate the potential for interactive effects of nitrogen and vitamin B12 and B1 starvation in eukaryotic phytoplankton. B12 plays essential roles in amino acid and one-carbon metabolism, while B1 is important for primary carbohydrate and amino acid metabolism and likely useful as an anti-oxidant. Here we will focus on three potential metabolic interconnections between vitamin, nitrogen, and sulfur metabolism that may have ramifications for the role of vitamin and nitrogen scarcities in driving ocean productivity and species composition. These include: (1) B12, B1, and N starvation impacts on osmolyte and antioxidant production, (2) B12 and B1 starvation impacts on polyamine biosynthesis, and (3) influence of B12 and B1 starvation on the diatom urea cycle and amino acid recycling through impacts on the citric acid cycle. We evaluate evidence for these interconnections and identify oceanographic contexts in which each may impact rates of primary production and phytoplankton community composition. Major implications include that B12 and B1 deprivation may impair the ability of phytoplankton to recover from nitrogen starvation and that changes in vitamin and nitrogen availability may synergistically impact harmful algal bloom formation.

Iron Limitation of a Springtime Bacterial and Phytoplankton Community in the Ross Sea: Implications for Vitamin B12 Nutrition

The Ross Sea is home to some of the largest phytoplankton blooms in the Southern Ocean. Primary production in this system has previously been shown to be iron limited in the summer and periodically iron and vitamin B12 colimited. In this study, we examined trace metal limitation of biological activity in the Ross Sea in the austral spring and considered possible implications for vitamin B12 nutrition. Bottle incubation experiments demonstrated that iron limited phytoplankton growth in the austral spring while B12, cobalt, and zinc did not. This is the first demonstration of iron limitation in a Phaeocystis antarctica-dominated, early season Ross Sea phytoplankton community. The lack of B12 limitation in this location is consistent with previous Ross Sea studies in the austral summer, wherein vitamin additions did not stimulate P. antarctica growth and B12 was limiting only when bacterial abundance was low. Bottle incubation experiments and a bacterial regrowth experiment also revealed that iron addition directly enhanced bacterial growth. B12 uptake measurements in natural water samples and in an iron fertilized bottle incubation demonstrated that bacteria serve not only as a source for vitamin B12, but also as a significant sink, and that iron additions enhanced B12 uptake rates in phytoplankton but not bacteria. Additionally, vitamin uptake rates did not become saturated upon the addition of up to 95 pM B12. A rapid B12 uptake rate was observed after 13 min, which then decreased to a slower constant uptake rate over the next 52 h. Results from this study highlight the importance of iron availability in limiting early season Ross Sea phytoplankton growth and suggest that rates of vitamin B12 production and consumption may be impacted by iron availability.

Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB

The alkane hydroxylase AlkB ofPseudomonas putidaGPo1 is typical of a large class of membrane-spanning diiron oxygenases that catalyze hydroxylation, epoxidation, and desaturation reactions. These enzymes are of considerable interest due to their impact on global hydrocarbon metabolism, their potential for practical biocatalytic application, and the resulting inspiration for the design of synthetic biomimetic catalysts. Although the three-dimensional structures of AlkB or any closely related proteins are unknown, topology modeling has predicted a structure comprised of six membrane-spanning helices with the catalytic iron diad appended to the cytoplasmic termini of the helix bundle. M(ssbauer data and alanine scanning have suggested that the diiron binding site is histidine-rich, as found in hemerythrin, and in contrast to the predominantly carboxylate binding motifs found in the diiron hydroxylases sMMO and T4MOh. Through protein side-chain mutations, a long, hydrophobic substrate-binding channel within the bundle has been identified that is tuned to accept medium-length alkanes. AlkB was the first alkane hydroxylase shown to generate a longlived substrate carbon radical during catalysis, as revealed by diagnostic skeletal rearrangements of the hydrocarbon probe norcarane. Herein we report results for the AlkB hydroxylation reaction using a panel of radical-clock substrates that display intrinsic rearrangement rates spanning five orders of magnitude, from a moderately slow 2.8 0 10 s 1 for bicyclo[3.1.0]hexane to an ultrafast 10 s 1 for trans-1-methyl-2phenylcyclopropane. Significantly, the ratios of rearranged and unrearranged products (R/U) found for the three mostslowly rearranging substrates were all in the range of unity even though their rearrangement rates differed widely. To account for these unusual results we propose a new diffusional model of AlkB hydroxylation that involves radical cagelike active-site dynamics of the type observed for hemeand cobalamin-containing metalloproteins. AlkB from P. putida GPo1 was expressed in P. putida GPo12 in the manner we have previously described. GPo12 is a receptacle clone that has been stripped of its innate hydroxylases and dehydrogenases. This approach has the advantages of producing unambiguous protein expression and high activity for only the inserted hydroxylase gene, while showing otherwise negligible background oxidation. Substrates were oxidized in resting whole cells and in cell-free extracts because all attempts to isolate and purify AlkB to date have led to loss of activity. Results for the AlkBmediated oxygenation of the three alkane substrates, bicyclo[4.1.0]heptane (norcarane, 1), bicyclo[3.1.0]hexane (2), and bicyclo[2.1.0]pentane (3), are presented in Table 1. These simple alkanes were chosen because of their similar size, nearly spherical shape, highly analogous structures, and similar rearrangement chemistry (Scheme 1). The data for all three substrates showed large amounts of rearrangement products (> 50%) consistent with the involvement of discreet radical intermediates during the hydroxylation process. Further, the ratios of primary to secondary alcohols formed from norcarane and bicyclohexane are similar to the partition ratios observed for bona fide radical reactions for these substrates (ca. 2 and 10%, respectively). It is striking, however, that the ratios of rearranged products to unrearranged products (R/U) for these three substrates do not correlate with the 100-fold change in the radical rearrangement rate constants for bicyclo[2.1.0]pent-2yl (kr = 20 10 9 s ), 2-norcaranyl (kr = 2 0 10 8 s ), and bicyclo[3.1.0]hex-2-yl (kr = 2.8 0 10 7 s ). We found the average R/U values for the three substrates remarkably constant (1.6, 1.6, and 4.7), corresponding to apparent radical lifetimes of 0.78, 7.8, and 170 ns, respectively. Indeed, bicyclohexane, with the slowest rearrangement rate, displayed the most rearranged product and by far the longest radical lifetime. The same effect was observed when norcarane and bicyclohexane were oxidized as a mixture. By contrast, the ultrafast rearranging probe trans-1-methyl-2-phenylcyclopropane (kr = 10 11 s ) was confirmed to afford only rearranged products. Clearly, there is a discrepancy here between the observed results for the more-slowly rearranging substrates and expectations based on Arrhenius-type kinetic behavior [*] D. Deng, R. S. Buzdygon, Prof. J. T. Groves Department of Chemistry, Princeton University Princeton NJ 08544 (USA) Fax: (+1)609-258-0348 E-mail: raustin@bates.edu

Identity and mechanisms of alkane-oxidizing metalloenzymes from deep-sea hydrothermal vents

Six aerobic alkanotrophs (organism that can metabolize alkanes as their sole carbon source) isolated from deep-sea hydrothermal vents were characterized using the radical clock substrate norcarane to determine the metalloenzyme and reaction mechanism used to oxidize alkanes. The organisms studied were Alcanivorax sp. strains EPR7 and MAR14, Marinobacter sp. strain EPR21, Nocardioides sp. strains EPR26w, EPR28w, and Parvibaculum hydrocarbonoclasticum strain EPR92. Each organism was able to grow on n-alkanes as the sole carbon source and therefore must express genes encoding an alkane-oxidizing enzyme. Results from the oxidation of the radical-clock diagnostic substrate norcarane demonstrated that five of the six organisms (EPR7, MAR14, EPR21, EPR26w, and EPR28w) used an alkane hydroxylase functionally similar to AlkB to catalyze the oxidation of medium-chain alkanes, while the sixth organism (EPR92) used an alkane-oxidizing cytochrome P450 (CYP)-like protein to catalyze the oxidation. DNA sequencing indicated that EPR7 and EPR21 possess genes encoding AlkB proteins, while sequencing results from EPR92 confirmed the presence of a gene encoding CYP-like alkane hydroxylase, consistent with the results from the norcarane experiments.

Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis

Almost all cells require thiamin, vitamin B1 (B1), which is synthesized via the coupling of thiazole and pyrimidine precursors. Here we demonstrate that 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cHET) is a useful in vivo B1 precursor for representatives of ubiquitous marine picoeukaryotic phytoplankton and Escherichia coli – drawing attention to cHET as a valuable exogenous micronutrient for microorganisms with ecological, industrial, and biomedical value. Comparative utilization experiments with the terrestrial plant Arabidopsis thaliana revealed that it can also use exogenous cHET, but notably, picoeukaryotic marine phytoplankton and E. coli were adapted to grow on low (picomolar) concentrations of exogenous cHET. Our results call for the modification of the conventional B1 biosynthesis model to incorporate cHET as a key precursor for B1 biosynthesis in two domains of life, and for consideration of cHET as a microbial micronutrient currency modulating marine primary productivity and community interactions in human gut-hosted microbiomes.