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Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

Isabelle Mary, Glen A. Tarran, Phillip E. Warwick, Matthew J. Terry, David J. Scanlan, Peter H. Burkill, Mikhail V. Zubkov
DOI: http://dx.doi.org/10.1111/j.1574-6941.2007.00414.x 36-45 First published online: 1 January 2008

Abstract

35S-Methionine and 3H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.

Keywords
  • marine phytoplankton
  • microbial photoheterotrophy
  • amino acid transport
  • flow cytometric sorting
  • dual tracer labelling

Introduction

Planktonic prokaryotes or bacterioplankton are the most numerous and biogeochemically important organisms of the open ocean (Kirchman, 1997; Ducklow, 2000). To date, three different groups of unicellular planktonic phototrophic bacteria, i.e. capable of harvesting visible sunlight energy or photosynthetically available radiation, have been described – aerobic oxygenic cyanobacteria (Waterbury et al., 1979; Chisholm et al., 1988), aerobic anoxygenic bacteria (Kolber et al., 2000, 2001) and proteorhodopsin-containing bacteria (Beja et al., 2000). None of these microorganisms are obligate photoautotrophs but rather photoheterotrophs with even the marine Prochlorococcus and Synechococcus cyanobacteria capable of taking up organic nitrogen, for example amino acids (Zubkov et al., 2003; Zubkov & Tarran, 2005).

There are several recent reports on the fact that visible sunlight stimulates bacterioplankton production in the open ocean. For example, uptake of leucine by the whole bacterioplankton community from the North Pacific Gyre, where Prochlorococcus are abundant, was 50–115% higher in light incubations than in dark incubations (Church et al., 2004, 2006). Similarly, uptake of leucine by total bacterioplankton was stimulated 3–60% by visible light in North Atlantic waters (Michelou et al., 2007). In these cited studies, large additions of leucine (20 nM) were administered to saturate transport and to minimize de novo synthesis of leucine in order to assess bacterioplankton production (Kirchman et al., 1986). However, artificially high concentrations of added leucine could allow acquisition by bacterioplankton cells with transport systems of lower affinity than that required in situ. Therefore, close to ambient concentrations of added tracer molecules would give a more realistic assessment of physiological responses of bacterioplankton cells to light in oligotrophic environments.

The effect of UV sunlight radiation on bacterioplankton groups has been recently investigated (Alonso-Saez et al., 2006). The aim of the present study was to assess the effect of visible light on the absolute rates of amino acid uptake by the dominant populations of bacterioplankton inhabiting oceanic surface waters. It has been hypothesized that light enhancement of uptake of amino acids at ambient concentrations could differ for the dominant microbial groups. A combined methodological approach of a bioassay of microbial uptake rates at ambient amino acid concentrations and flow cytometric sorting of isotopically labelled bacterioplankton cells was employed to examine this hypothesis. Because of the insurmountable difficulties in recreating realistic environmental conditions for oligotrophic microorganisms in laboratory culture, the experimental work was carried out in the field, on two meridional transatlantic cruises. The cruises provided access to different oligotrophic areas of the central Atlantic Ocean, where the effect of light on the microbial uptake of amino acids was expected to be the most pronounced. Amino acids were chosen as model molecules because their concentration in oligotrophic waters is below 1 nM, they are taken up by the majority of microbial cells, and cells require energy to transport them and incorporate them into proteins. Also, bacterioplankton groups could selectively take up individual amino acids.

Materials and methods

Sampling site

Experimental work was performed during two meridional transatlantic oceanographic cruises, on board the Royal Research Ship James Clark Ross (Cruise No. JR91) in September–October 2003, and on board the Royal Research Ship Discovery (Cruise No. D299) in October–November 2005 (Fig. 1a). At every station, surface seawater samples were collected from a depth of 2–7 m with a sampling rosette of 20-L Niskin bottles mounted on a conductivity-temperature-depth (CTD) profiler to determine bacterioplankton abundance and microbial uptake rates of amino acids. Parallel light and dark incubations of samples inoculated with isotopic tracers were performed at six stations on the 2003 cruise and eight stations on the 2005 cruise (Fig. 1a). Prochlorococcus and three to four other bacterioplankton groups were preloaded with 35S-methionine (2003 cruise) and with 3H-leucine and 35S-methionine (2005 cruise), and were then flow cytometrically sorted. This study focusses on the analysis of these light/dark experiments.

1

Schematic presentation of the central Atlantic Ocean (a), studied on two meridional transect cruises in October 2003 and November 2005. Triangles indicate the main stations, at which dark and light uptake rates of amino acids by microbial groups were determined. Solid lines with white dots indicate cruise tracks and nonessential stations, at which dark–light experiments were not done. Thick dashed lines indicate the boundaries of the main oceanic provinces (b) Latitudinal distribution of surface water temperature and salinity in the two main study areas. Large inverted triangles indicate the main stations.

Bioassay of amino acid concentration and microbial uptake rates using radioactively labelled precursors

The bioavailable concentrations and turnover rates of amino acids were estimated using isotope dilution series (Wright & Hobbie, 1966; Zubkov & Tarran, 2005). Briefly, the samples used for rate determinations were initially collected into acid-washed 1-L thermos flasks using acid-soaked silicone tubing and processed within 1 h after sampling. The l-[35S]methionine (specific activity >37 TBq mmol−1) was added at a standard concentration of 0.1 nM and diluted with nonlabelled methionine using a dilution series of 0.2, 0.5, 1.0, 1.5 and 2.0 nM. The l-[4,5-3H]leucine (specific activity 6 TBq mmol−1) was added in a series of 0.1, 0.2, 0.4, 0.6, 0.8 nM final concentration. Triplicate samples (1.6 mL) for each amino acid addition were incubated in 2 mL sterile, clear polypropylene microcentrifuge tubes (Starlab, Milton Keynes, UK) with screw caps in the dark at in situ temperatures. One sample was fixed at 10, 20 and 30 min, respectively, by adding 20% paraformaldehyde to 1% (v/w) final concentration. The sample particulate material was harvested onto polycarbonate filters with pore size 0.2 μm. Radioactivity retained on filters was measured as disintegrations per minute (DPM) using liquid scintillation counters (Tri-Carb 3100TR, Perkin-Elmer, Beaconsfield, UK) onboard ships. The rate of precursor uptake was calculated as the slope of the linear regression of radioactivity against incubation time for each amino acid concentration in a series. These rates were used to estimate maximum ambient bioavailable concentrations of amino acids and their microbial uptake rates under an assumption that microbial affinity is negligibly small compared to natural concentrations of amino acids (Zubkov & Tarran, 2005). The short, 10 min incubations were used to reduce the effect of potential metabolism of labelled amino acids and the release of labelled metabolites on the estimates of net microbial uptake rates. The net uptake rates were shown to closely correlate with rates of precursor assimilation in microbial macromolecules, e.g. proteins (Zubkov et al., 1998).

Determination of bacterioplankton abundance

Seawater samples of 1.8 mL were fixed with paraformaldehyde 1% final concentration. Prochlorococcus and Synechococcus cyanobacteria were enumerated in unstained samples with a FACSort flow cytometer (Becton Dickinson, Oxford, UK) using their specific chlorophyll/phycoerythrin autofluorescence (Olson et al., 1993). Abundance of total bacterioplankton (Fig. 2a and b) and Prochlorococcus, in cases where its chlorophyll fluorescence was below the detection limit, was determined after staining with SYBR Green I DNA dye (Marie et al., 1997). Three main bacterioplankton groups were visualized, based on their DNA content and light scatter properties (Fig. 2c and d): first, a cluster of cells with low nucleic acid (LNA) content; second, a heterogeneous cluster contained cells with high nucleic acid (HNA) and low 90° light scatter or side scatter (HNA-ls); and the third cluster contained cells with HNA and high light scatter (HNA-hs), which is dominated by Prochlorococcus in oligotrophic surface waters (Fig. 2c). Yellow–green 0.5 μm reference beads (Fluoresbrite Microparticles, Polysciences, Warrington) were used in all analyses as an internal standard for both fluorescence and flow rates. The absolute concentration of beads in the stock solution was determined using syringe pump flow cytometry (Zubkov & Burkill, 2006).

2

Characteristic flow cytometric density plot signatures of SYBR Green – DNA stained bacterioplankton (Bpl) of (a) oligotrophic and (b) mesotrophic surface waters. Flow-sorted clusters of LNA containing cells, HNA containing cells with low 90° light scatter (HNA-ls), HNA containing cells with high 90° light scatter (HNA-hs), and Prochlorococcus (Pro) cells separated in (c) oligotrophic and (d) mesotrophic areas. Polygon regions are drawn around the Bpl cluster. Arrows indicate the clusters of sorted cells and singlet reference beads.

Phylogenetic characterization of cells, flow sorted from the dominant bacterioplankton groups in the studied areas, using FISH, have been reported elsewhere (Mary et al., 2006; Zubkov et al., 2007). These showed remarkable similarity of group composition in the North and South Atlantic. FISH analyses were carried out at four of the 14 stations under current analysis.

Flow cytometric sorting of radioactively labelled bacterioplankton groups

Amino acid uptake of bacterioplankton groups was determined using 35S-methionine single labelling at 0.85 nM on the 2003 cruise (Zubkov & Tarran, 2005) or 3H-leucine (0.8 nM) and 35S-methionine (0.5 nM) dual labelling on the 2005 cruise. The chosen amino acid concentrations were required for detectable labelling of the flow-sorted cells. The concentrations were higher than the ambient amino acid concentrations (Fig. 3) but still within the range that did not stimulate disproportional microbial uptake, which was assessed using the amino acid concentration series (‘Bioassay of amino acid concentration and microbial uptake rates using radioactively labelled precursors’) of the same samples.

3

Latitudinal distribution of the dominant microbial groups, bioavailable amino acids and their microbial uptake rates in surface waters of the main areas studied. (a) The abundance of total and LNA bacterioplankton, (b) the abundance of Prochlorococcus (Pro) and Synechococcus (Syn) cyanobacteria, (c) bioavailable concentrations and total microbial uptake rates of methionine (Met) (d) bioavailable concentrations and total microbial uptake rates of leucine (Leu). Large inverted triangles indicate the main stations. Dotted lines connect the measurements made at adjoining stations. Error bars show single SDs (a, b) and single SEs (c, d) of respective measurements. Vertical dashed lines indicate the position of the southern subtropical front.

Five replicate 1.6 mL samples were incubated in a laboratory, air conditioned at the surface water in situ temperature (temperature was monitored using a digital thermometer, immersed in water) in the dark (in a rack wrapped in aluminium foil) and at visible sunlight on a window sill. The glass window blocked UV radiation and reduced visible light by 4%. The polypropylene crystal clear microcentrifuge tubes (Starlab, Milton Keynes, UK), in which the samples were incubated, transmitted 72% of light at 400 nm, increasing approximately linearly to 82% at 700 nm. Therefore, except for the absence of UV light, the light conditions of the incubated microbial cells were comparable with the light conditions at the sampled depth of c. 7 m. On the 2003 cruise, the samples, incubated in the dark and light for 3 h, were fixed with 1% paraformaldehyde at 2 °C for 24 h and stored frozen at −80 °C before being analysed ashore (Zubkov & Tarran, 2005). On the 2005 cruise, the samples were incubated in a similar way but were incubated for 2 h, also fixed with 1% paraformaldehyde at 2 °C. However, on the latter cruise the flow sorting of bacterioplankton groups was conducted on board the ship within the next 12–36 h.

The groups of stained bacterioplankton (Fig. 2) were flow sorted, using single-cell sort mode, at a rate of 10–250 particles s−1. Prochlorococcus and Synechococcus (2003 cruise only, the two most southerly stations) cyanobacteria were sorted from unstained samples (Zubkov & Tarran, 2005). Sorted cells were collected onto 0.2 μm pore size nylon filters, washed with deionized water and radioassayed. Three to four proportional numbers of the cells (from 1, 2, 3 and 4 × 103 cells to 10, 20, 30 and 40 × 103 cells, depending on cell concentration in a sample) were sorted, and the mean cellular tracer uptake was determined as the slope of the linear regression of radioactivity against the number of sorted cells. Radioactivity retained on filters was provisionally assayed using a standard counter and accurately measured as counts per minute (CPM) using an ultra-low-level liquid scintillation counter (1220 Quantulus, Wallac, Finland). The latter counter uses a logarithmic analogue to digital converter, which effectively expands the low energy end of the spectrum and facilitates more effective deconvolution of the two spectra in the case of dual labelled cells. Tritium and 35S counting efficiencies and spill-over of the 35S into the 3H counting window were corrected for, using a quench-dependent calibration. Subsequently, DPM were calculated to correct for the radioactive decay.

Sorting purity was assessed routinely by budgeting radioactivity of flow-sorted cells from different clusters (Zubkov & Tarran, 2005) as well as by sorting one type of bead from a mixture of two 0.5 μm beads (Zubkov et al., 2007). The sorted material was 99% enriched with the target particles; the sorted particle recovery was >95%.

For comparison between stations, the absolute uptake rates of Prochlorococcus and LNA cells were calculated using the estimated total microbial uptake rates of amino acids at ambient concentrations (Fig. 3c and d). The total microbial uptake rate equals the uptake rate of a population of flow-sorted average bacterioplankton cells (Zubkov et al., 2004) and the sum of the uptake rates of all dominant bacterioplankton populations. The uptake of Prochlorococcus and LNA populations mL−1 of seawater was calculated by multiplying the average cell uptake of tracer by the abundance of cells, determined by flow cytometry. The population uptake was divided by the uptake of total bacterioplankton, calculated by multiplying the uptake of an average bacterioplankton cell by the abundance of bacterioplankton. The computed fractions of the Prochlorococcus and LNA population were multiplied by the absolute rates of microbial uptake of methionine or leucine to calculate the absolute rates of amino acid uptake by the respective populations. The latter rates were divided by Prochlorococcus and LNA cell abundance and multiplied by the number of molecules in 1 mol (Avogadro constant) to estimate the absolute cell uptake rates of amino acids.

Results

Microbial abundance and activity in the studied area

The effect of light on amino acid uptake was tested in two areas of the central Atlantic Ocean (Fig. 1a) – the central–southern part of the South Atlantic gyre, flanked by the south subtropical frontal zone, and the southern part of the North Atlantic gyre, adjoining the Equatorial convergence zone. The physical parameters of the surface waters were different in the two areas (Fig. 1b). A coincidental meridional decrease in surface water temperature and salinity characterized the southern area, studied on the 2003 cruise. A pronounced decrease in salinity and high temperature characterized the northern area, studied on the 2005 cruise. Total bacterioplankton abundance was generally lower in the southern area, although the concentration of bacterioplankton rose sharply towards the frontal zone (Fig. 3a). The LNA group comprised 43±4% of total bacterioplankton irrespective of the area, confirming earlier observations of the constancy of relative abundance of the LNA bacteria in central Atlantic waters (Mary et al., 2006). Prochlorococcus comprised 27±4% of the total bacteria in the northern area and 19±1% in the central part of the southern area with a dramatic drop to 1% in the frontal zone (Fig. 3b). Conversely, the absolute abundance of Synechococcus in the frontal zone increased 10 times and their cells represented 6% of total bacterioplankton in that zone, compared with 1–2% to the north of it. Although the physical environment and abundance of bacterioplankton in the two studied areas were different, the relative abundance of the LNA group and Prochlorococcus were similar, with the exception of the frontal zone, where Prochlorococcus was replaced by Synechococcus.

The changes in concentrations and the daily total microbial uptake of methionine and leucine were remarkably synchronized in both areas studied, except for the frontal zone (Fig. 3c and d). A pronounced peak of all parameters was observed in the convergence zone. Leucine concentrations were always considerably lower than the daily uptake of leucine, pointing to turnover of leucine within 2–27 h, while the concentrations and daily uptake of methionine were similar with a corresponding slower turnover of methionine within 4–56 h. As a result, bacterioplankton abundance and the total microbial uptake of amino acids were generally higher in the northern area than in the southern area, with the exception of the frontal zone. Consequently, the two areas complemented each other and, when combined, they gave a good range of conditions for general assessment of the effect of light on the bacterioplankton transport of amino acids at ambient subnanomolar concentrations (Fig. 3c and d).

The effect of light on the uptake rate of amino acids by the dominant bacterioplankton groups

The uptake of methionine and leucine by total bacterioplankton was 22% higher (t-test: paired two-sample for means of the combined data set for the two amino acids, P<0.001), when incubated in the light compared to their uptake in the dark (Fig. 4a). Similarly, the uptake of methionine and leucine by an average flow-sorted bacterioplankton cell was 24% higher (t-test, P<0.001) in the light than in the dark (Fig. 4b). The comparisons indicated that bacterioplankton cells increase their uptake of amino acids in the light but it is not clear as to whether the cells were responding similarly or whether only one or several groups of cells responded to light. To answer this question the authors flow sorted different groups of bacterioplankton (Fig. 2c and d).

4

Scatter plot comparisons of methionine (Met) and leucine (Leu) uptake by total bacterioplankton, presented as a percentage of added radioactivity, retained by particulate material (Total Bpl, a) and flow-sorted average bacterioplankton (Avg. Bpl, b) cells in the dark vs. light incubations of surface water samples, collected at the main stations on the 2003 (03) and 2005 (05) cruises. The dashed line is a unity line. Error bars show single SEs of measurements. The figure legend is the same for (a) and (b).

Prochlorococcus cells showed a 50% increase (t-test, P<0.001) in the uptake of methionine and leucine in the light, compared with dark incubations (Fig. 5a), the highest increase for the groups studied. The uptake of both amino acids by LNA cells was 23% higher (t-test, P<0.003) in the light (Fig. 5b). In the same experiments the uptake of amino acids by the HNA-ls, HNA-hs, excluding Prochlorococcus, as well as by the combined HNA cells, excluding Prochlorococcus, did not differ significantly (t-test, P>0.3) in the dark and light incubations (Fig. 5c). The uptake of methionine by Synechococcus cells was 140% higher under light in the two experiments, conducted in the frontal zone, where these cyanobacteria were sufficiently numerous for flow sorting without preconcentration of cells (Fig. 5c), but no generalizations are made based on this small dataset.

5

Scatter plot comparisons of methionine (Met) and leucine (Leu) uptake by Prochlorococcus cells (Pro, a), LNA containing cells (LNA, b) and HNA containing cells with low (HNA-ls) and high (HNA-hs excluding Pro cells) 90° light scatter in the dark vs. light incubations of surface water samples, collected at the main stations on the 2003 (03) and 2005 (05) cruises. Asterisks indicate estimates, calculated for the whole HNA cell cluster (high and low light scatter) by subtracting the sum of Pro and LNA populations from the total bacterioplankton uptake of amino acids. The dashed line is a unity line. Error bars show single SEs of measurements.

The results showed that in general, light enhanced the uptake rates of amino acids by the Prochlorococcus and LNA cells. In order to assess whether any other major groups of bacterioplankton were missing, whose amino acid uptake could be systematically enhanced by light, the contributions of the Prochlorococcus and LNA populations to the total light enhancement were budgeted. By multiplying the average 50% light enhancement of a Prochlorococcus cell by the average 23% of Prochlorococcus abundance relative to total bacterioplankton abundance in the studied oligotrophic areas, 12% contribution of the Prochlorococcus population to light enhancement of total bacterioplankton is obtained. By multiplying the average 23% light enhancement of a LNA cell by the average 43% of LNA abundance relative to total bacterioplankton abundance in the studied oligotrophic areas, 10% contribution of the LNA population to light enhancement of total bacterioplankton is obtained. The 22% sum of the two contributions equals the determined 22% light enhancement of total bacterioplankton. Therefore, there was no other main bacterioplankton population, in which amino acid uptake was enhanced by light. However, the above calculation is based on average values, indicating a general trend and does not exclude specific cases, where amino acid uptake by Prochlorococcus or LNA cells is not stimulated by light or the uptake by other groups could be enhanced by light, as the scattering of data points in Fig. 5 suggests.

Absolute uptake rates of amino acids by the dominant bacterioplankton groups

The results, presented in Fig. 5, although being the original data, can only be compared as dark and light pairs, because absolute rates of cell uptake at different stations depended on the concentration of amino acids in the seawater. Similar to relative values (Fig. 5a and b), the absolute rates of the Prochlorococcus and LNA cells were higher in the light incubations (Fig. 6a and b).

6

Scatter plot comparisons of calculated absolute methionine (Met) and leucine (Leu) uptake rates by Prochlorococcus cells (Pro, a) and LNA containing cells (LNA, b) in the dark vs. light incubations of surface water samples, collected at the main stations on the 2003 (03) and 2005 (05) cruises. The figure legend is the same for (a) and (b). (c) Corresponding scatter plot comparison of calculated absolute leucine (Leu) vs. methionine (Met) uptake rates by Pro and LNA cells. The dashed line is a unity line. Error bars show single SEs of measurements.

Using average values and SDs to assist comparisons, LNA cells acquired 12 300±11 400 methionine molecules cell−1 h−1 in the dark vs. 16 300±17 500 methionine molecules cell−1 h−1 in the light incubations and 29 300±11 700 leucine molecules cell−1 h−1 in the dark vs. 36 200±20 400 leucine molecules cell−1 h−1 in the light incubations. Prochlorococcus cells acquired 16 000±11 700 methionine molecules cell−1 h−1 in the dark vs. 27 300±19 000 methionine molecules cell−1 h−1 in the light incubations, and 21 100±11 500 leucine molecules cell−1 h−1 in the dark and 31 600±13 500 leucine molecules cell−1 h−1 in the light incubations. The above rates show that the two cell populations compared here took up comparable amounts of amino acids. Light increased the uptake of methionine and leucine by LNA cells by 4 × 103 and 7 × 103 molecules cell−1 h−1, respectively, while the uptake of both methionine and leucine by Prochlorococcus cells was increased in the light by 11 × 103 molecules cell−1 h−1, showing that the uptake of the latter cells was enhanced by light more than the uptake of the former. Therefore, Prochlorococcus cells seem to exploit sunlight for amino acid uptake more efficiently than LNA cells.

Discussion

Light enhancement of amino acid uptake by bacterioplankton groups

This observational study was designed to detect the phenomenon of light enhanced amino acid uptake. The total microbial uptake of methionine in the dark ranged between 0.15 and 1.3 nmol L−1 day−1, to which the LNA population contributed 20–60%, the Prochlorococcus population contributed 8–50% in the oligotrophic Atlantic and the Synechoccocus population contributed 2% in the frontal zone. The total microbial uptake of leucine in the dark ranged between 0.2 and 1.7 nmol L−1 day−1, to which the LNA population contributed 30–60% and the Prochlorococcus population contributed 7–40%. When exposed to visible light the LNA cells on average increased amino acid uptake by 23% and Prochlorococcus cells by 50%. Under visible light the total microbial uptake of amino acids on average increased by 22%. The authors' observations corroborate studies in other oceanic regions (Church et al., 2004, 2006; Michelou et al., 2007) and highlight the necessity of taking into account the effect of light on bacterioplankton metabolism and production.

Explanation of the mechanism for powering transmembrane transport by light will require further study. A plausible working hypothesis is that the photosynthetic apparatus of Prochlorococcus cells harvests light and transfers this energy into ATP that is used for additional powering of the active uptake of amino acids against the steep gradient of subnanomolar concentrations found in seawater. Ecologically this would seem to be a very efficient way of utilizing an abundant light energy source for amino acid acquisition in an ultimately nitrogen-depleted environment. A similar strategy could also be used by LNA cells. Sixty per cent of the LNA cells that were identified in the studied samples are Pelagibacter or SAR11-related cells (Mary et al., 2006; Zubkov et al., 2007), which are likely to possess proteorhodopsin (Giovannoni et al., 2005a). Although proteorhodopsin presence has not been shown in the flow-sorted LNA cells in the present study, a large proportion of SAR11 cells appear to have this photoreceptor (Campbell et al., 2007). In this case, light energy could be directly harvested by the proteorhodopsin proton pump (Beja et al., 2001). These pumps have been shown to be active in Pelagibacter cells although no light induction was observed in culture (Giovannoni et al., 2005a), possibly because of the insufficiently nutrient-depleted conditions modelled in the laboratory. This field study has shown that light could be utilized by SAR11 cells to enhance amino acid transport by an average of 23%. If it is taken into account that SAR11 cells constitute 60% of the LNA cells and speculate that the remaining 40% of cells are not capable of harvesting light, then the actual light enhancement of amino acid uptake by SAR11 could increase upto 38%, which is only 12% less than light enhancement by Prochlorococcus cells, which are larger in size.

Keeping in mind that LNA cells are twice as abundant as Prochlorococcus cells (Fig. 3a and b), the light stimulated increase in amino acid uptake of these two populations is similar, 10% and 12%, respectively. The sum of their contributions accounts for the whole 22% light increase of amino acid uptake by the total bacterioplankton community. In other words, in the studied regions of the Atlantic Ocean (Fig. 1a) there were no other bacterioplankton populations, whose amino acid uptake was increased by light. In contrast Michelou (2007) reported 40% unaccountable light stimulation of amino acid uptake in bacterioplankton communities in the North Atlantic Ocean. It is possible that other groups of bacterioplankton not studied by the authors, e.g. the LNA group, could be stimulated by light in temperate waters and higher latitudes. What is encouraging is that light stimulation of cyanobacteria was observed in both studies. The results showing light stimulation of amino acid uptake in LNA cells are more controversial. Alonso-Saez (2006) reported sunlight, including UV light, inhibited leucine uptake but not ATP uptake by SAR11 cells in Mediterranean coastal waters in spring. Therefore, there could be a differential response of these cells to light depending on the nutrient regime encountered as well as the light quality, e.g. UV light exposure that requires further investigation.

Comparison of methionine and leucine uptake rates

The uptake rates of amino acids varied within approximately one order of magnitude, from 3 × 103 to 70 × 103 molecules cell−1 h−1. This is the first estimate of absolute values for uptake of these amino acids and so comparison to other studies is not possible. However, the present values are within the range of estimates, uncorrected for ambient amino acid concentrations, of cell uptake of methionine in the suboxic waters of the Arabian Sea (Zubkov et al., 2006).

Tracing the uptake of two amino acids into bacterioplankton cells simultaneously, using dual labelling, offered a unique comparison of the absolute uptake rates of the two amino acids. Differences in uptake rates of methionine and leucine by Prochlorococcus and LNA cells observable in Fig. 6a and b becomes apparent when the uptake rates of the two amino acids are plotted against each other (Fig. 6c). Prochlorococcus cells have similar uptake rates for methionine and leucine, while LNA cells show a preference for leucine. Prochlorococcus cells take up on average 21 600±16 500 methionine molecules cell−1 h−1 and 26 300±13 200 leucine molecules zcell−1 h−1, which are statistically similar values (t-test with equal variance, P>0.1). The LNA cells take up on average 14 300±9300 methionine molecules cell−1 h−1 and 32 700±16 400 leucine molecules cell−1 h−1, which are statistically different values (t-test with unequal variance, P<0.001).

The observations are remarkable in their own right because it shows that different groups of bacterioplankton can selectively take up organic molecules, present in waters at subnanomolar concentrations (Fig.3c and d). Presently, there is insufficient data to explain why the uptake rate of methionine is 82% of the rate of leucine uptake by Prochlorococcus cells, whereas methionine uptake is only 44% of the leucine uptake in LNA cells. Why does Prochlorococcus require more methionine than LNA cells? It is unlikely that proteins of Prochlorococcus cells are enriched in methionine compared with proteins of LNA cells. It is more plausible that Prochlorococcus could use methionine not only as an amino acid, like LNA cells, but also as a source of reduced sulphur, for example, for synthesis of sulpholipids, which are abundant in Prochlorococcus cell membranes (Van Mooy et al., 2006). The observed differences in amino acid uptake rates could also mean that the LNA cells preferentially transport leucine at ambient concentrations consistently lower than the concentrations of methionine (Fig. 3c and d). Compared with methionine, leucine is a more common amino acid present in proteins and bacterial cells may need to transport more leucine than methionine for direct protein synthesis. Prochlorococcus has only a limited number of genes encoding membrane transporters (Rocap et al., 2003) that could be used for selective uptake of molecules essential for its metabolism, e.g. reduced sulphur sources. On the other hand, compared to any other bacteria whose genomes have been sequenced, Pelagibacter has about 60% more genes encoding membrane transporters (Giovannoni et al., 2005b), mainly high affinity ATP-binding cassette transporters. Therefore, genetically the Pelagibacter related LNA cells could be more adapted for efficient transport of amino acids in proportions required for direct protein synthesis.

Thus, the 35S-methionine and 3H-leucine tracer experiments have proven the usefulness of a new technique, which is based on flow cytometric sorting of dual labelled natural bacterioplankton cells. Using this method, it was observed that visible sunlight could provide energy to enhance uptake of amino acids at ambient concentrations. Groups of bacteria with HNA content showed no light stimulation. Two numerically dominant groups of oceanic bacterioplankton, Prochlorococcus cyanobacteria and bacteria with LNA content, comprised of 60% SAR11-related cells, on average increase their uptake of amino acids in the visible light by 50% and 23%, respectively, compared with dark incubations. The populations of these two bacterioplankton cell populations completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community. To the authors' knowledge this is the first evidence that the SAR11 enriched group of oligotrophic bacterioplankton could increase uptake of amino acids under visible light.

Acknowledgements

The authors gratefully acknowledge the captain, officers, crew and fellow scientists aboard RRS James Clark Ross and RRS Discovery for their help during the two cruises. This work formed a part of the Atlantic Meridional Transect (AMT) Consortium programme (NER/O/S/2001/00680) and was supported by a small grant (NE/C514723/1) of the Natural Environment Research Council (NERC), UK, by the NERC Marine Microbial Metagenomics consortium (NE/C50800X/1) and by the National Oceanography Centre and Plymouth Marine Laboratory Core Programmes. The research of M.V.Z. was supported by the NERC advanced research fellowship (NER/I/S/2000/01426). This is an AMT contribution No. 160.

Footnotes

  • Editor: Michael Wagner

References

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