James W. Ammerman

Phosphorus deficiency in the Atlantic: An emerging paradigm in oceanography

Nitrogen, iron, and silica are widely considered to be the most important nutrients that limit phytoplankton growth in the worlds oceans. Though clearly important in lakes, the role of phosphorus has been largely ignored in the ocean. In part, this is because of early studies that suggested there was excess phosphate (P) relative to the needs of the phytoplankton in open ocean waters. Thanks to recent studies at the Hawaiian Ocean Time (HOT) series station (Station ALOHA) in the North Pacific subtropical gyre [Karl et al., 2001, and references therein], there is a growing appreciation of the potential importance of phosphorus as a limiting nutrient in subtropical Pacific waters.

The alkaline phosphatase PhoX is more widely distributed in marine bacteria than the classical PhoA

Phosphorus (P) is a vital nutrient for all living organisms and may control the growth of bacteria in the ocean. Bacteria induce alkaline phosphatases when inorganic phosphate (Pi) is insufficient to meet their P-requirements, and therefore bulk alkaline phosphatase activity measurements have been used to assess the P-status of microbial assemblages. In this study, the molecular basis of marine bacterial phosphatases and their potential role in the environment were investigated. We found that only a limited number of homologs to the classical Escherichia coli alkaline phosphatase (PhoA) were present in marine isolates in the Bacteroidetes and γ-proteobacteria lineages. In contrast, PhoX, a recently described phosphatase, was widely distributed among diverse bacterial taxa, including Cyanobacteria, and frequently found in the marine metagenomic Global Ocean Survey database. These taxa included ecologically important groups such as Roseobacter and Trichodesmium. PhoX was induced solely upon P-starvation and accounted for approximately 90% of the phosphatase activity in the model marine bacterium Silicibacter pomeroyi. Analysis of the available transcriptomic datasets and their corresponding metagenomes indicated that PhoX is more abundant than PhoA in oligotrophic marine environments such as the North Pacific Subtropical Gyre. Those analyses also revealed that PhoA may be important when Bacteroidetes are abundant, such as in algal bloom episodes. However, PhoX appears to be much more widespread. Its identification as a gene that mediates organic P acquisition in ecologically important groups, and as a marker of Pi-stress, constitutes an important step toward a better understanding of the marine P cycle.

A Review of Water Column Processes Influencing Hypoxia in the Northern Gulf of Mexico

In this review, we use data from field measurements of biogeochemical processes and cycles in the Mississippi River plume and in other shelf regions of the northern Gulf of Mexico to determine plume contributions to coastal hypoxia. We briefly review pertinent findings from these process studies, review recent mechanistic models that synthesize these processes to address hypoxia-related issues, and reinterpret current understanding in the context of these mechanistic models. Some of our conclusions are that both nitrogen and phosphorus are sometimes limiting to phytoplankton growth; respiration is the main fate of fixed carbon in the plume, implying that recycling is the main fate of nitrogen; decreasing the river nitrate loading results in less than a 1:1 decrease in organic matter sinking from the plume; and sedimenting organic matter from the Mississippi River plume can only fuel about 23% of observed coastal hypoxia, suggesting significant contributions from the Atchafalaya River and, possibly, coastal wetlands. We also identify gaps in our knowledge about controls on hypoxia, and indicate that some reinterpretation of our basic assumptions about this system is required. There are clear needs for improved information on the sources, rates, and locations of organic matter sedimentation; for further investigation of internal biogeochemical processes and cycling; for improved understanding of the rates of oxygen diffusion across the pycnocline; for identification and quantification of other sources of organic matter fueling hypoxia or other mechanisms by which Mississippi River derived organic matter fuels hypoxia; and for the development of a fully coupled physical-biogeochemical model.

Molecular response of the bloom-forming cyanobacterium, Microcystis aeruginosa, to phosphorus limitation.

Cyanobacteria blooms caused by species such as Microcystis have become commonplace in many freshwater ecosystems. Although phosphorus (P) typically limits the growth of freshwater phytoplankton populations, little is known regarding the molecular response of Microcystis to variation in P concentrations and sources. For this study, we examined genes involved in P acquisition in Microcystis including two high-affinity phosphate-binding proteins (pstS and sphX) and a putative alkaline phosphatase (phoX). Sequence analyses among ten clones of Microcystis aeruginosa and one clone of Microcystis wesenbergii indicates that these genes are present and conserved within the species, but perhaps not the genus, as phoX was not identified in M. wesenbergii. Experiments with clones of M. aeruginosa indicated that expression of these three genes was strongly upregulated (50- to 400-fold) under low inorganic P conditions and that the expression of phoX was correlated with alkaline phosphatase activity (p < 0.005). In contrast, cultures grown exclusively on high levels of organic phosphorus sources (adenosine 5′-monophosphate, β-glycerol phosphate, and d-glucose-6-phosphate) or under nitrogen-limited conditions displayed neither high levels of gene expression nor alkaline phosphatase activity. Since Microcystis dominates phytoplankton assemblages in summer when levels of inorganic P (Pi) are often low and/or dominate lakes with low Pi and high organic P, our findings suggest this cyanobacterium may rely on pstS, sphX, and phoX to efficiently transport Pi and exploit organic sources of P to form blooms.

Role of the phosphatase PhoX in the phosphorus metabolism of the marine bacterium Ruegeria pomeroyi DSS-3

Marine microbes are adapted to surviving in a variable phosphorus (P) environment. This adaptation frequently involves the presence of periplasmic or cell membrane-associated enzymes that enable them access to alternative sources of P when phosphate is depleted. In a recent study we identified the phosphatase PhoX as an enzyme that may be essential in mediating organic P acquisition in the ocean. Here we have investigated the role of this enzyme in the utilization of different P sources, using as a model the marine bacterium Ruegeria pomeroyi DSS-3. Although our previous study had demonstrated that PhoX accounts for more than 90% of the alkaline phosphatase (APase) activity in R. pomeroyi, a PhoX mutant strain was able to grow on monophosphate esters at the same rate as the wild type. Nevertheless, further APase kinetic analyses with both strains demonstrated that the Km of the wild-type strain was an order of magnitude lower than the mutant strain, indicating that PhoX is crucial for the use of these substrates at low concentrations, typically found in seawater. We also showed that PhoX is required for efficient hydrolysation of nucleotides like ADP and ATP.

Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser-induced fluorescence detection†

Microbial ectoenzyme activities in aquatic environments are important determinants of polymer hydrolysis and indicators of the state of microbial carbon, nitrogen, and phosphorus nutrition. Marine ectoenzymes are found on the cell surface or in the periplasmic space of gram‐negative heterotrophic bacteria. Phosphatases, which remove phosphate groups from substrates, are one example of an ectoenzyme. Enzyme assays based on‐capillary electrophoresis (CE) take advantage of CEs high‐efficiency separation, extremely low sample volume requirements, and its ability to electrophoretically mix and separate zones of enzymes, substrates, and products all in one experimental run. CE has better resolving power and, when utilized with laser‐induced fluorescence (LIF) detection, it is more sensitive than chromatography. CE‐LIF is a promising tool for determining different phosphatases within a single microbial strain as well as the functional diversity between strains. In this study, four bacterial strains were studied (Shewanella sp., TW7, BB2AT2, and Vibrio alginolyticus) with each yielding at least one phosphatase that was kinetically characterized. Km values were calculated and found to be in the range of 0.0725–3.35 µM, whereas Vmax values ranged from 1.02×10−3 to 1.05×10−2 µM/min. The large range of values demonstrates differences among the phosphatases, suggesting different roles for each phosphatase not only between the species but also within a single bacterial species. This can have the important implications for organic matter processing in the sea.