Although cyanobacteria are unable to incorporate some organic compounds, e.g., thymidine (10), Synechococcus axenic cultures utilize urea (7, 21) and amino acids (5, 19, 23) at a low rate and even show aminopeptidase activity (18). However, Synechococcus photoheterotrophy has always been considered ecologically unimportant. Current knowledge about Prochloroccocus nutrient assimilation (24) is limited, partly because of the existence of only one axenic Prochloroccocus culture (26). Interestingly, this strain (PCC9511) was isolated in the presence of an amino acid, methionine. It was reported that Prochlorococcus cultures take up NH₄⁺ but that they cannot utilize NO₃⁻ or NO₂⁻; they can also use urea but not other sources of organic nitrogen (21, 26), although amino acids were not tested as a sole nitrogen source.

Genomic data from the draft annotations of the MIT9313 and MED4 strains of Prochlorococcus spp. show that they possess several transporter systems for amino acids, as shown on the Department of Energy Joint Genome Institute website (http://www.jgi.doe.gov); however, a further analysis is necessary for the prediction of possible utilization pathways. The genome of the MED4 strain lacks the nitrate and nitrite reductase genes, a further proof that this strain needs to rely on reduced nitrogen compounds, e.g., NH₄⁺, amino acids, etc.

In working with laboratory cultures of cyanobacteria, it is very challenging to simulate oceanic oligotrophic conditions and the results of laboratory nutrient addition experiments can be extrapolated only with considerable caution. On the other hand, studies done with natural microbial communities may reveal roles of dominant groups, including cyanobacteria, more directly and unambiguously. Therefore, we chose a field study in which the ³⁵S-labeled methionine tracer was used as a proxy for labile nitrogen-containing compounds. The present study is probably the first that demonstrates the role of Prochlorococcus in the consumption of organic, nitrogen-containing compounds in the oligotrophic oceanic ecosystem.

The samples (1.6 ml) were inoculated with l-[4,5-³H]leucine (specific activity, 171 Ci/mmol) at a 5 nM final concentration or l-[³⁵S]methionine (specific activity, >1,000 Ci/mmol) at an ~1 nM final concentration and incubated in the dark at the in situ temperatures. The subsamples (0.5 ml) were fixed with 1% paraformaldehyde (PFA) at 0.5, 1, and 1.5 h. The samples (33 ml) were inoculated with l-[U-¹⁴C]leucine (295 mCi/mmol) at a 20 nM final concentration. The subsamples (10 ml) were fixed at 0.5, 1.0, and 1.5 h by being mixed with an equal volume of 10% trichloroacetic acid (TCA). The sample particulate materials were harvested onto 0.2-μm-pore-size nylon filters (Pall Corporation, Ann Arbor, Mich.). Radioactivity retained on the filters was measured as disintegrations per minute with a RackBeta 1409 liquid scintillation counter (LKB-Wallac, Turku, Finland). The rate of precursor incorporation was calculated as the slope of the linear regression of radioactivity against the incubation time (r² > 0.96, P < 0.0001) (Fig. ​(Fig.1a).1a). The time series experiments showed that the bacterioplankton had low nonspecific absorption of precursors. Uptake or incorporation rate constants, i.e., fractions of the added precursors consumed by the bacterioplankton per day, were used for rate comparison.

The metabolic activities of bacterioplankton groups, including cyanobacteria, were determined by using the consumption rate of [³⁵S]methionine (34). Methionine was added at an ~1 nM concentration to determine approximate in situ rates of amino acid turnover. Four replicated samples were incubated at in situ temperature in the dark, fixed with 1% PFA after 3 h of incubation, and kept frozen. Thawed samples were stained with SYBR Green I (see above), and visualized bacterioplankton groups and whole communities were flow sorted by using FACSort and FACSCalibur flow cytometers (Becton Dickinson), both of which were set to single-cell sort mode at a sorting rate of 10 to 150 particles s⁻¹. Deionized water (18 MΩ) was used as a sheath fluid to enhance the washing of sorted PFA-fixed cells. Cyanobacteria were identified in the stained samples by using their red autofluorescence. Sorted cells were collected onto 0.2-μm-pore-size nylon filters, washed with deionized water, and radioassayed (see above). Three proportional numbers of the cells (e.g., 1 × 10³, 2 × 10³, and 3 × 10³ to 10 × 10³, 20 × 10³, and 30 × 10³ cells, respectively) were sorted, and the cellular metabolic activities were determined as the slopes of the linear regressions of radioactivity against the numbers of sorted bacteria (Fig. ​(Fig.1b).1b). The reproducibility of gate sorting was checked with six samples, which were sorted in duplicate on different days or by using different instruments (Fig. ​(Fig.1b).1b). The determined [³⁵S]methionine cellular uptake correlated with a 1:1 relationship (r² = 0.99, P < 0.0001, n = 6).

Prochlorococcus spp. were very abundant in waters from the equator to 8°N [(3.0 ± 1.6) × 10⁸ cells liter⁻¹] and were later replaced at the northern end of the transect by Synechococcus spp. [(1.0 ± 0.5) × 10⁸ cells liter⁻¹ ]. The clear switching of dominance between these two taxa coincided with a transition from the oligotrophic region, characterized by the primary production of 180 ± 16 mg of C m⁻² day⁻¹ (number of samples, 4) to the mesotrophic region with primary production of 330 ± 56 mg of C m⁻² day⁻¹ (number of samples, 4) (G. R. C. Morgan and A. P. Rees, personal communication). In the oligotrophic region, Prochlorococcus comprised 96.5% ± 3% of all cyanobacteria, while Synechococcus clearly dominated the mesotrophic region, accounting for 97% ± 5% of all cyanobacteria.

We found that both Prochlorococcus and Synechococcus cells incorporated the precursor at a rate comparable to the rates of average bacterioplankton cells (Fig. ​(Fig.3a).3a). The mean cellular specific activity of a Prochlorococcus cell was 110% of the activity of an average bacterial cell in the oligotrophic region, while the mean activity of a Synechococcus cell was only 30% of the activity of an average bacterial cell in the mesotrophic region. The methionine turnover rate constants of both cyanobacteria suggested that these might decrease with depth (Fig. ​(Fig.3b3b).

Although both Prochlorococcus and Synechococcus cyanobacteria were able to incorporate dissolved amino acids at close to natural concentrations, the ecological significance of this differed widely between the two cyanobacterial genera (Fig. ​(Fig.3).3). In the mesotrophic region, Synechococcus consumed on average only 1.5% of the dissolved methionine pool per day, compared to an average 46% daily turnover by the other bacterioplankton; in other words, Synechococcus was responsible for only 3% of methionine turnover by all bacterioplankton (Fig. ​(Fig.3c).3c). Conversely, in oligotrophic waters Prochlorococcus turned over on average 8% of the methionine pool daily, compared to 16% turned over daily by the other bacterioplankton, or 33% of the methionine turnover by all bacterioplankton, showing that it could compete directly with other bacterioplankton populations. The competitive success of Prochlorococcus was spectacular, since it was able to consume one-third of the amino acid pool and represent one-third of the abundance of the total bacterioplankton community. Considering that the contribution of Prochlorococcus to total phytoplankton production could be as high as 50% (15), Prochlorococcus is undoubtedly the key algal species in oceanic oligotrophic ecosystems.

Assuming that in the oligotrophic region Prochlorococcus contributed about 50% (15) of the total phytoplankton primary production of 180 mg of C m⁻² day⁻¹, and with a 6.6 C/N molar ratio (25), the Prochlorococcus population nitrogen demand should be about 1.1 mM N m⁻² day⁻¹. The leucine-derived bacterioplankton production was estimated to be 25 ± 9 mg of C m⁻² day⁻¹(number of samples, 4) in the photic zone of the oligotrophic region (M. V. Zubkov, unpublished data), to which Prochlorococcus contributed about 30% or 0.1 mM N m⁻² day⁻¹. Therefore, the dissolved amino acids, which are an energetically preferential nitrogen source, were estimated to satisfy about 10% of the Prochlorococcus requirements for nitrogen, while ammonium may have satisfied the remainder of their nitrogen requirements. By consuming amino acids, Prochlorococcus deprived other bacteria of an important source of organic nitrogen, forcing the latter to use inorganic nitrogen at a higher energetic cost in the organic-carbon-limited oligotrophic environment.

Such strong competition for nutrients may be the driving force behind a shift of photoheterotrophic bacteria towards photosynthesis (4, 14). It was reported that the contribution of these bacteria to total photosynthesis rises from about 1% in nutrient-rich regions to 10% in oligotrophic regions (13).

The presented results may help to explain the current controversy in balancing the carbon cycle in the oligotrophic central regions of the ocean with low primary production (31), where community respiration may exceed photosynthesis (8). The dominance of Prochlorococcus, which consumes organic as well as inorganic nitrogen pools, may generate about a 30% overestimation of bacterial secondary production and hence of computed organic matter decomposition and respiration in oligotrophic waters.