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Applied and Environmental Microbiology, May 2007, p. 3028-3033, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02606-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Lack of Control of Nitrite Assimilation by Ammonium in an Oceanic Picocyanobacterium, Synechococcus sp. Strain WH 8103{triangledown}

Michael Wyman* and Clare Bird

School of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom

Received 8 November 2006/ Accepted 23 February 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In cyanobacteria, the transcriptional activator NtcA is involved in global nitrogen control and, in the absence of ammonium, regulates the expression of genes involved in the assimilation of alternative nitrogen sources. The oceanic picocyanobacterium Synechococcus sp. strain WH 8103 harbors a copy of ntcA, but in the present study, we show that unlike other marine cyanobacteria that have been investigated, this strain is capable of coassimilating nitrite when grown in the presence of ammonium. Transcript levels for the genes encoding the nitrate/nitrite-bispecific permease NrtP and nitrate reductase (NarB) were substantially down-regulated by ammonium, whereas the abundances of nitrite reductase (NirA) transcripts were similar in nitrite- and ammonium-grown cells. The growth of Synechococcus sp. strain WH 8103 in medium containing both ammonium and nitrite resulted in only minor changes in the expression profile in comparison to that of nitrite-grown cells with the exception that the gene encoding the high-affinity ammonium transporter Amt1 was down-regulated to the levels seen in ammonium-grown cells. Whereas the expression of nrtP, narB, and amt1 appears to be NtcA dependent in this marine cyanobacterium, the transcription and expression of nirA appear not to be. The ability to coassimilate nitrite and reduced-nitrogen sources like ammonium may be an adaptive trait that enables oceanic strains like Synechococcus sp. strain WH 8103 to exploit the low nitrite concentrations found in oceanic surface waters that are not available to their principal and more numerous competitor, Prochlorococcus.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Picocyanobacteria of the genera Synechococcus and Prochlorococcus dominate the photosynthetic biomass found in the surface waters of subtropical and tropical oceans, where they contribute substantially to global carbon fixation (4, 18, 22, 27). The majority of Synechococcus spp. are capable of utilizing oxidized forms of combined nitrogen (nitrate and nitrite), whereas the majority of Prochlorococcus spp. cannot (17). Those that can are able to utilize nitrite rather than nitrate and are found exclusively among the low-light-adapted ecotypes that have been isolated from the base of the euphotic zone. By contrast, the high-light-adapted ecotypes from nutrient-poor surface waters all rely on reduced forms of nitrogen exclusively and therefore cannot utilize either nitrate or nitrite.

The spatial distribution of these two groups of marine picocyanobacteria overlaps across a gradient of nutrient availability (18). Synechococcus spp. thrive in mesotrophic and moderately oligotrophic waters where they are able to exploit both oxidized and reduced forms of nitrogen. At the other extreme, Prochlorococcus spp. are much more abundant in the most highly stratified, nutrient-poor waters, where they all but displace Synechococcus spp. as the dominant picocyanobacterial group. The predominance of Prochlorococcus spp. in highly stratified waters suggests that they are more effective scavengers of regenerated forms of nitrogen such as ammonium than Synechococcus spp. This is perhaps to be expected since by virtue of their smaller size, Prochlorococcus spp. have a significantly higher surface-area-to-volume ratio and, due to differences in their light-harvesting apparatus (chlorophyll a2/b2 versus phycobilisomes), somewhat lower cell N requirements (25).

Notwithstanding these considerations, the comparatively rapid division rates reported for both groups of picocyanobacteria in surface waters dominated by Prochlorococcus spp. (18, 22) suggest that neither group is particularly severely nitrogen limited. Therefore, this begs an intriguing question. How are Synechococcus spp. able to successfully coexist with Prochlorococcus spp. (albeit in considerably lower numbers) even though they have a higher cell nitrogen quota and are apparently inferior in their capacity to exploit low concentrations of ammonium nitrogen? One possible explanation is that Synechococcus spp. may be more efficient at utilizing alternative forms of regenerated nitrogen, but in the case of amino acids at least, this appears not to be the case (29). Another possibility is that in addition to competing for ammonium, Synechococcus spp. may be able to exploit the very low concentrations of oxidized nitrogen present in the upper water column that are generally unavailable to Prochlorococcus spp.

Depth profiles of combined nitrogen concentrations from the upper mixed layer of highly oligotrophic waters show a primary nitrite maximum associated with the top of the pycnocline that occurs above the main nitracline. The concentrations of nitrate and nitrite in surface waters, like ammonium, are in the nanomolar range but may be less than an order of magnitude lower (2). At such low ambient concentrations, it seems unlikely that ammonium would negatively regulate the assimilation of nitrate or nitrite to any great extent, but making predictions about its effect on the expression of nitrogen assimilation genes is complicated by the quite variable responses reported for picocyanobacteria (1, 11, 12, 20, 21).

Much is known about the nitrogen control of N assimilation in freshwater cyanobacteria, in which it has been shown that ammonium represses the expression of genes required for the utilization of alternative forms of combined nitrogen such as nitrate and nitrite and, in diazotrophs, dinitrogen (5, 9). In the absence of ammonium, the expression of permease genes and those encoding the structural components of nitrate reductase (narB), nitrite reductase (nirA), glutamine synthetase (glnA), and (in diazotrophs) nitrogenase (nifHDK) is activated. Activation requires the transcription factor NtcA, which binds to a conserved motif (GTAN8TAC) 22 nucleotides upstream of the –10 region of N-regulated promoters. A second transcription factor, NtcB, is required for the full transcriptional activation of some genes including the nir operon of Synechococcus sp. strain PCC 7942 (24).

Nitrogen control in marine cyanobacteria has been investigated to a far lesser extent, and while there are parallels to freshwater strains, there are also some intriguing differences. In the coastal strain Synechococcus sp. strain PCC 7002, ammonium represses the expression of nrtP (a nitrate/nitrite permease) and narB in a manner that is very similar to that found in freshwater cyanobacteria (21). Both genes have a canonical NtcA-regulated promoter located upstream of their coding regions, which, in the case of nrtP, has been mapped to the transcriptional start point (21). Ammonium rapidly represses ntcA expression in the marine Synechococcus sp. strain WH 7803 at concentrations above 1 µmol liter–1 (13, 14) and completely inhibits the uptake of nitrate from the growth medium (8, 13).

The inhibitory effects of ammonium on nitrite assimilation are less immediate in Synechococcus sp. strain WH 7803 such that uptake rates decline gradually over a period of 24 h following the addition of ammonium (12). Nevertheless, the capacity to utilize both nitrate and nitrite is absolutely dependent upon activation by NtcA in this strain since neither nitrogen source can support growth of an ntcA mutant, whereas growth on ammonium is unaffected (20), nor can this mutant degrade biliproteins in response to N deprivation, a phenotype that is also shared by ntcA null mutants of freshwater cyanobacteria. In comparison to nrtP and narB, nirA appears to be only moderately down-regulated by ammonium in the oceanic strain Synechococcus sp. strain WH 8103 (1). It has a weak NtcA-like promoter with the architecture GTAN8AAC, which primer extension experiments revealed directs nirA expression in N-starved cells, but this could not be demonstrated in N-replete cells. Nitrogen control of nirA, therefore, appears to be more relaxed in this marine strain than that reported for freshwater cyanobacteria.

In the present study, we show that Synechococcus sp. strain WH 8103 is capable of utilizing nitrite even when grown in the presence of comparatively high ammonium concentrations. By means of quantitative reverse transcriptase PCR (QRT-PCR), we confirm and extend our previous semiquantitative results regarding the differential effects of ammonium on the expression of narB and nirA and other genes involved in nitrogen assimilation. We put forward the hypothesis that the lack of tight ammonium regulation of nirA expression is an adaptive trait in oceanic Synechococcus spp. that may highlight the underlying importance of nitrite utilization in their production ecology. Coutilization of nitrite and ammonium might explain why oceanic Synechococcus spp. are not subject to complete competitive exclusion by Prochlorococcus spp. in warm, highly stratified, oligotrophic waters.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture conditions.
Replicate experimental cultures (n = 3) of Synechococcus sp. strain WH 8103 were grown at 25°C under constant irradiance (20 µmol photons m–2 s–1) in ASW medium (28) containing either NaNO3 (2 mM), NaNO2 (0.9 mM), NH4Cl (1.1 mM), or a combination of 1.1 mM (or 0.11 mM) NH4Cl and 0.9 mM NaNO2 as nitrogen sources. For experimental media containing NaNO2, cultures were inoculated with cells growing on NH4Cl rather than NaNO3 as a nitrogen source and then subcultured at least twice prior to experimentation. This preconditioning treatment was found to be necessary to prevent the rapid collapse of NaNO2 subcultures when inoculated from media containing NaNO3 (see below). Growth was determined by measuring the optical density of cell suspensions at 750 nm (OD750).

Isolation of RNA and QRT-PCR.
Cell suspensions of mid-logarithmic-phase cells were pelleted at 4°C (8,000 x g) for 5 min. Total RNA was isolated with the RiboPure-Bacteria kit (Ambion, Inc.) according to the manufacturer's instructions and including a final DNase I treatment at 30°C for 30 min. First-strand cDNA synthesis using 0.5 µg of RNA as a template was carried out in a total volume of 20 µl using the RT primer mix and reagents contained within the QuantiTect reverse transcription kit (QIAGEN Ltd.). Following elimination of any residual DNA by incubating RNA preparations for 5 min at 42°C with the gDNA Wipeout buffer supplied with the kit, the cDNA synthesis reaction was carried out at 42°C for 15 min in duplicate reactions with or without QuantiTect reverse transcriptase, followed by a 3-min incubation at 95°C to inactivate the enzyme.

Gene internal primers for seven target genes (Table 1) were designed using previously published gene sequences from Synechococcus sp. strain WH 8103 (1) (GenBank accession numbers AAG42270 [amt1], ABC02233 [ntcA], and AAB84115 [glnA]) or the closely related strain WH 8102 (accession number Q7U524 [rnpB]). Optimal primer concentrations were established empirically, and their specificities were verified in control reactions containing equal quantities of genomic DNA and by subsequent DNA sequencing of the products. Quantitative PCR was carried out with each primer pair at a concentration of 12 pmol each with 0.5 µl of the cDNA synthesis reactions (or the no-reverse-transcriptase controls) using the Brilliant SYBR green QPCR Master Mix kit and the Mx3000p real-time PCR thermocycler and fluorescence detection system (Stratagene). PCRs were carried out in 96-well plates in 25-µl volumes following the activation of the SureStart Taq DNA polymerase included in the kit at 95°C for 10 min. The cycling conditions were as follows: 95°C for 30 s, 60°C for 60 s, and 72°C for 60 s for 40 cycles, followed by a single cycle at 95°C for 30 s, 55°C for 30 s, and a ramp to 95°C at 0.2°C s–1 to determine the melting temperature of the amplified cDNA and to check for the absence of secondary products.


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  		TABLE 1. Oligonucleotide primers used for QRT-PCR

 
Data handling procedures.
The relative abundance of individual mRNAs within a particular sample was normalized to that of the constitutively expressed gene rnpB (see, e.g., reference 3) using the equation Formula, where CT unknown and CT rnpB are the threshold cycles (CTs) for detection of the test (e.g., narB) and control (rnpB) cDNAs, respectively. In practice, this was a relatively minor normalization procedure, since the amount of RNA used in the cDNA reactions was similar for all N treatments but does correct for variability in instrument sensitivity and the fluorescence threshold assigned to the CT between experiments. The equation assumes a similar amplification efficiency for each target gene (i.e., a doubling in the abundance of the product with each cycle). The equal amplification efficiencies of the primer pairs were established during primer optimization experiments using genomic DNA as a template (CT = 19.58 ± 0.41; n = 7), which also confirmed that each gene was present as a single (or equal) copy on the genome.

The normalized abundance of an mRNA species detected in experimental nitrogen treatments compared to that in ammonium-grown cells was calculated as follows: Formula. The output of this equation expresses how much more or less abundant (n-fold) an individual mRNA species is than that in ammonium-grown cells.

Determination of nutrient concentrations.
Samples for the determination of nutrient concentrations were obtained by pelleting cells at 13,000 x g for 2 min at 25°C and removing an aliquot of the culture medium above the cell pellet. Nitrite concentrations were determined by a standard colorimetric technique (23) using a dilution series of NaNO2 in N-free ASW medium to generate the standard curve. Ammonium concentrations were determined using the reagents described previously by Holmes et al. (10) rather than the standard indophenol method. In our hands, the indophenol method produced unacceptably high and variable background readings that we suspect may be attributable to interference from the Tris-HCl in ASW medium (standards made up in seawater were not affected). The ammonium method described previously by Holmes et al. is a high-sensitivity fluorometric technique, but for the less exacting purposes of the present investigation, we determined ammonium concentrations by measuring the absorption at 360 nm following color development rather than fluorescence emission. Standards were prepared in N-free ASW medium with a dilution series of NH4Cl and showed a linear increase in A360 between the lower limit of detection (0.015 mM) and 2 mM NH4Cl (r2 = 0.999).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of nitrogen source on transcript abundance of nitrogen assimilation genes.
The normalized CTs detected for cDNAs synthesized from total RNA from ammonium-grown cells were significantly higher for nrtP (encoding a nitrate/nitrite permease), narB (encoding nitrate reductase), and ntcA (encoding a transcriptional activator involved in nitrogen control in cyanobacteria). Hence, the mRNAs for these genes are somewhat less abundant in Synechococcus sp. strain WH 8103 grown with ammonium as the sole nitrogen source than they were in the alternative sources of combined nitrogen examined (Fig. 1). The comparatively lower CTs detected for these three genes and for cDNAs derived from nirA (encoding nitrite reductase) and amt1 mRNA (encoding an ammonium transporter) (16) indicate that these four genes are up-regulated in nitrate-grown cells. This was confirmed by normalizing transcript abundance to that detected in ammonium-grown cells, and this showed that the concentrations of mRNAs derived from nrtP, narB, nirA, ntcA, and amt1 were approximately 50-, 21-, 4.5-, 4.6-, and 4-fold more abundant, respectively, in nitrate-grown cells (Fig. 2).


Figure 1
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  		FIG. 1. Abundance of transcripts from six different nitrogen assimilatory genes in Synechococcus sp. strain WH 8103 grown in media containing different nitrogen sources and determined by QRT-PCR. The CT value is the CT at which cDNAs were first detected during amplification. For a given gene, if the CT is higher in one treatment than in another, this indicates that the mRNA in the starting RNA preparation was present at a lower concentration. The log10 mean CT values ± 1 standard deviation (SD) (n = 3) plotted are normalized to the mean CT of rnpB and show how much higher (>1) or lower (<1) the CT of a cDNA is in comparison to that determined for this constitutively expressed gene in each treatment. The primers designed and used in this study amplify all target genes with equal efficiencies.

 

Figure 2
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  		FIG. 2. Mean relative abundances (±1 standard deviation [SD]) (n = 3) of transcripts in cells grown in different nitrogen sources normalized to that determined for the same gene in cells grown with ammonium as the sole nitrogen source. The data show how much more abundant (n-fold) (where abundance equals >1) or, conversely, less abundant (where abundance equals <1) a particular mRNA is in each treatment. For example, where abundance equals 50, that mRNA is 50-fold more abundant than in ammonium-grown cells, whereas where the abundance is 0.5, that mRNA is 2-fold less abundant.

 
By contrast, when grown in the presence of nitrite, the abundances of transcripts derived from nrtP and narB were only 3.8- and 3.0-fold higher, respectively, than those from ammonium-grown cells, and those from ntcA and amt1 were approximately 3.0- and 1.7-fold higher, respectively, while nirA mRNA was present at a similar concentration (Fig. 2). The gene expression profile for cells grown in the presence of a combination of nitrite and ammonium was similar to that of nitrite-grown cells with the exception of amt1 mRNA, which was present at a concentration similar to that in ammonium-grown cultures. The abundance of transcripts from glnA (encoding glutamine synthetase) showed the least variability with nitrogen source (Fig. 2). This result contrasts with that reported previously (1), which showed that glnA mRNA was more abundant in ammonium-grown cells, and demonstrates the value of normalizing to rnpB to correct for global effects on transcription in cells growing at slightly different growth rates.

Utilization of mixed combined nitrogen sources during growth.
The QRT-PCR experiments reported above showed that growth in the presence of nitrate resulted in the significant up-regulation of both nrtP and narB in comparison to their up-regulation in ammonium-grown cells. By contrast, the presence of ammonium either singly or in combination with nitrite had comparatively little effect on the abundance of nirA mRNA. To ascertain whether Synechococcus sp. strain WH 8103 retains the ability to utilize nitrite in the presence of ammonium, the growth of cultures and the assimilation of nitrite and ammonium were investigated using media containing nitrite at 0.9 mM and ammonium at two different concentrations (0.11 mM and 1.1 mM).

Initial growth rates were similar in both media (µ = 0.5 to 0.6 day–1) but declined thereafter as culture turbidity increased (Fig. 3 and 4). Growth in media containing the lower ammonium concentration was supported almost exclusively by nitrite until the time point at which this nitrogen source had declined to about 50% of the starting concentration (Fig. 3). After this, both nitrite and ammonium were taken up simultaneously until they were both exhausted by ~200 h after the start of the experiment.


Figure 3
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  		FIG. 3. Growth curve of Synechococcus sp. strain WH 8103 in media containing nitrite and low concentrations of ammonium. Log10 mean OD750 values ± 1 standard deviation (SD) (open triangles), mean ammonium concentrations ± 1 SD (open circles), and mean nitrite concentrations ± 1 SD (filled circles) are shown (n = 3).

 

Figure 4
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  		FIG. 4. Growth curve of Synechococcus sp. strain WH 8103 in media containing nitrite and high concentrations of ammonium. Log10 mean OD750 values ± 1 standard deviation (SD) (open triangles), mean ammonium concentrations ± 1 SD (open circles), and mean nitrite concentrations ± 1 SD (filled circles) are shown (n = 3). Note the different scale for the x axis in comparison to that in Fig. 3.

 
To test whether a higher concentration of ammonium might repress nitrite utilization more effectively, the starting ammonium concentration was increased by an order of magnitude to 1.1 mM. As was found in the above-described experiment, nitrite was utilized preferentially during the initial part of the experiment and was exhausted by ~200 h (Fig. 4). Approximately equal quantities of ammonium and nitrite were coassimilated once the latter had declined to less than 0.2 mM. After 200 h, growth was supported exclusively by ammonium, and this resulted in a further doubling in biomass. The mean biomass reached at the end of the experiment was >70% higher than that reached at the lower ammonium concentration, although in neither case had growth ceased completely.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The quantitative data reported here add further support to our previous observations (1) and confirm that both narB and nrtP are substantially up-regulated in nitrate-grown cells of Synechococcus sp. strain WH 8103 in comparison to those utilizing alternative nitrogen sources. A similar marked reduction in the abundance of both mRNAs in the presence of ammonium has been reported for the coastal isolate Synechococcus sp. strain PCC 7002 that is concomitant with a rapid decline in nitrate reduction (21). As has been demonstrated in this strain, Synechococcus sp. strain WH7803 (12), and a variety of freshwater cyanobacteria, it therefore appears unlikely that this oceanic strain is capable of assimilating nitrate in the presence of ammonium (assuming that, as in these cases, transcript levels correlate with gene expression and activity).

Interestingly, neither gene is up-regulated to the same extent in nitrite-grown cells. Therefore, the presence of nitrate in the growth medium may be required for the complete activation of narB and nrtP. When Synechococcus sp. strain WH 8103 is transferred to N-free medium, however, the abundances of both mRNAs are elevated above that in nitrate-grown cells within 8 h of N starvation (1). Clearly, then, the requirement for nitrate for the full activation of nrtP and narB is not absolute. Rather, the higher abundance of both transcripts, and nrtP mRNA in particular, may be symptomatic of the greater degree of N stress experienced by cells assimilating nitrate rather than alternative nitrogen sources. The higher abundance of ntcA mRNA in nitrate-grown cells is certainly consistent with this scenario.

Nitrate transport is the rate-limiting step for nitrate assimilation in cyanobacteria (6), and the up-regulation of nrtP appears to be critical for ensuring an adequate supply of nitrogen to nitrate-grown cells. An nrtP mutant strain of Synechococcus sp. strain PCC 7002 is capable of only slow growth on nitrate and displays symptoms of chlorosis even at high concentrations (21). By contrast, the assimilation of nitrite by this nrtP mutant is unaffected under normal growth conditions. NrtP is a nitrate/nitrite-bispecific permease (21), however, and therefore, it appears odd that the abundance of nrtP mRNA was an order of magnitude lower in nitrite-grown cells of Synechococcus sp. strain WH 8103.

Growth rates were similar on both nitrogen sources, but, seemingly, nitrite-grown cells are able to satisfy their nitrogen needs without the requirement to induce nrtP to the same extent as occurs with nitrate. Nitrite can enter the cell by passive diffusion as nitrous acid (7), however, and, in Synechococcus sp. strain PCC 7002 and the freshwater Synechococcus sp. strain PCC 7942, at least, via a second, as-yet-unidentified transporter (15, 21). Thus, nitrite-grown cells are likely to be less dependent than those utilizing nitrate on NrtP-mediated transport of the nitrogen source and appear to be able to satisfy their nitrogen requirements by up-regulating nrtP to a lesser extent. In fact, the low-level activation of nrtP compared to that in nitrate-grown cells may be critical since, in our hands, successful transfers of Synechococcus sp. strain WH 8103 to media containing nitrite could be achieved only with cultures growing with ammonium rather than nitrate. Nitrite is toxic at high concentrations, and the collapse of cultures transferred to nitrite from nitrate-containing media may be due to nitrite rapidly flooding the cell via NrtP-mediated transport before the concentrations of the transporter within the cell membrane can be adjusted.

Perhaps a further indicator of the greater nitrogen stress experienced by nitrate-grown cells is that amt1 was up-regulated above the basal levels observed in ammonium-containing media. Although ammonium was not added to the media containing nitrate, the up-regulation of this high-affinity transporter may enable nitrate-grown cells to scavenge ammonium lost to the external medium from the cytoplasm, as has been demonstrated in the freshwater strain Synechococcus sp. strain PCC7942 (26). If this interpretation is correct, nitrate-grown cells of Synechococcus sp. strain WH 8103 appear to maximize the utilization of the ammonium generated via the reduction of oxidized nitrogen to a greater extent than nitrite-grown cultures in which amt1 is up-regulated to a level less than twice that in ammonium-grown cells.

The abundance of amt1 mRNA was at approximately the same concentration as that in ammonium-grown cells when ammonium was supplied for growth in combination with nitrite. Apart from this subtle difference, however, the abundance of mRNAs encoding the other genes investigated in nitrate/ammonium-grown cells was very similar to that found in cells utilizing nitrite exclusively. In the case of nirA, transcript abundance was actually slightly higher (up to twofold) in ammonium-grown cells than it was in those provided with nitrite as the sole nitrogen source or in combination with ammonium. Therefore, the presence of ammonium in the growth medium had little influence on nirA expression in Synechococcus sp. strain WH 8103 when nitrite was also present.

It seemed possible, therefore, that Synechococcus sp. strain WH 8103 may be able to utilize nitrite in the presence of ammonium. This was investigated under an incident irradiance similar to that near the base of the euphotic zone in subtropical/tropical waters where nitrite concentrations are highest. While Synechococcus sp. strain WH 8103 was able to coassimilate both nitrogen sources, nitrite was used immediately following subculture. By contrast, the major utilization of ammonium occurred later in the growth cycle, even when ammonium was available at millimolar concentrations.

As occurs in freshwater cyanobacteria, nitrite assimilation declines and then ceases (albeit with a delay) following the addition of ammonium to Synechococcus sp. strain WH 7803, a strain that is typical of those found in shelf waters (12). Both nitrate and nitrite reductase activities decline rapidly following the transfer of Synechococcus sp. strain PCC 7002 from a nitrate-containing medium to one containing urea (21). As in media containing ammonium, urea completely represses the expression of both narB and nrtP in this coastal strain. However, whereas the negative effect of ammonium on the expression of these genes in Synechococcus sp. strain WH 8103 indicates that the effect on nitrate assimilation and reduction is likely to be similar to that found in these other marine Synechococcus spp., the growth experiments show that this does not apply to nitrite utilization. Why, then, is nitrogen regulation in this oceanic strain so different from that found in coastal and shelf water isolates?

Of the environmental variables that vary between the habitats occupied by marine Synechococcus spp., the most obvious is the considerably lower concentration of combined nitrogen found in open waters in comparison to that in waters located closer to land. Whereas Prochlorococcus spp. are found in far fewer numbers or are entirely absent from coastal and shelf waters (18), in the warm, nutrient-poor open oceans, Synechococcus spp. have to compete for nitrogen with both heterotrophic bacteria and these much more numerous picocyanobacteria. In the surface waters of these regions, the growth of Prochlorococcus spp. is supported entirely by ammonium and other reduced forms of nitrogen. Given the likely advantage that Prochlorococcus spp. enjoy by virtue of their smaller size, it is therefore surprising that Synechococcus spp. are able to maintain a foothold in the face of this additional competition.

In a recent study conducted in the Gulf of Aqaba, seasonally driven oscillations in the populations of these picocyanobacteria were associated with changes in the relative abundance of different genotypes of Synechococcus spp. throughout the year (19). Members of clade III (which includes strain WH 8103) were detected only during summer stratification, suggesting that this clade is specifically adapted to low-nutrient concentrations. If the capacity to coutilize ammonium and nitrite is widespread among members of clade III (and perhaps other low-nutrient-adapted clades), the ability to access the low concentrations of nitrite present in stratified waters may be an important adaptive trait since surface-water-dwelling populations of Prochlorococcus spp. are generally incapable of assimilating this source of combined nitrogen. While Synechococcus would incur additional energy costs for nitrite assimilation, there may be a tradeoff in effectively reducing competition for the limiting resource.


   ACKNOWLEDGMENTS
 
This research was supported by a studentship awarded to C.B. (GT04/97/280/MAS) and the Marine and Freshwater Biodiversity Programme funded by the Natural Environment Research Council of the United Kingdom.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom. Phone: 44 (0)1786 467784. Fax: 44 (0)1786 464994. E-mail: mw4{at}stir.ac.uk Back

{triangledown} Published ahead of print on 2 March 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bird, C., and M. Wyman. 2003. Nitrate/nitrite assimilation system of the marine picoplanktonic cyanobacterium Synechococcus sp. strain WH 8103: effect of nitrogen source and availability on gene expression. Appl. Environ. Microbiol. 69:7009-7018.[Abstract/Free Full Text]
  2. Brzezinski, M. A. 1988. Vertical distributions of ammonium in stratified oligotrophic waters. Limnol. Oceanogr. 33:1176-1182.
  3. Cheng, Y., J.-L. Li, L. Shi, L. Wang, A. Latifi, and C.-C. Zhang. 2006. A pair of iron-responsive genes encoding protein kinases with a Ser/Thr kinase domain are regulated by NtcA in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 188:4822-4829.[Abstract/Free Full Text]
  4. Chisolm, S. W., R. J. Olson, E. R. Zettler, R. Goericke, J. B. Waterbury, and N. A. Welschmeyer. 1988. A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature 334:340-343.[CrossRef]
  5. Flores, E., and A. Herrero. 1994. Assimilatory nitrogen metabolism and its regulation, p. 487-517. In D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  6. Flores, E., and A. Herrero. 2005. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem. Soc. Trans. 33:164-167.[CrossRef][Medline]
  7. Flores, E., A. Herrero, and M. G. Guerrero. 1987. Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta 896:103-108.
  8. Glibert, P. M., and R. T. Ray. 1990. Different patterns of growth and nitrogen uptake in two clones of marine Synechococcus spp. Mar. Biol. 107:273-280.[CrossRef]
  9. Herrero, A., A. M. Muro-Pastor, and E. Flores. 2001. Nitrogen control in cyanobacteria. J. Bacteriol. 183:411-425.[Free Full Text]
  10. Holmes, R. M., A. Aminot, R. Kérouel, B. A. Hooker, and B. J. Peterson. 1999. A simple and precise method for measuring ammonium in marine and freshwater ecosystems. Can. J. Fish Aquat. Sci. 56:1801-1808.[CrossRef]
  11. Lindell, D., D. Erdner, D. Marie, O. Prasil, M. Koblizek, F. Le Gall, R. Rippka, F. Partensky, D. J. Scanlan, and A. F. Post. 2002. Nitrogen stress response of Prochlorococcus strain PCC 9511 (Oxyphotobacteria) involves contrasting regulation of glnA and amt1. J. Phycol. 38:1113-1124.[CrossRef]
  12. Lindell, D., E. Padan, and A. F. Post. 1998. Regulation of ntcA expression and nitrite uptake in the marine cyanobacterium Synechococcus sp. strain WH 7803. J. Bacteriol. 180:1878-1886.[Abstract/Free Full Text]
  13. Lindell, D., E. Padan, and A. F. Post. 1999. Effect of ammonium on nitrate/nitrite uptake and ntcA expression in Synechococcus sp. strain WH 7803. Bull. Inst. Oceanogr. 19:273-278.
  14. Lindell, D., and A. F. Post. 2001. Ecological aspects of ntcA gene expression and its use as an indicator of the nitrogen status of marine Synechococcus spp. Appl. Environ. Microbiol. 67:3340-3349.[Abstract/Free Full Text]
  15. Maeda, S., M. Okamura, M. Kobayashi, and T. Omata. 1998. Nitrite-specific active transport system of the cyanobacterium Synechococcus sp. strain PCC 7942. J. Bacteriol. 180:6761-6763.[Abstract/Free Full Text]
  16. Montesinos, M. L., A. M. Muro-Pastor, A. Herrero, and E. Flores. 1998. Ammonium/methylammonium permeases of a cyanobacterium: identification and analysis of three nitrogen-regulated amt genes in Synechocystis sp. PCC 6803. J. Biol. Chem. 273:31463-31470.[Abstract/Free Full Text]
  17. Moore, L. R., A. F. Post, G. Rocap, and S. W. Chisolm. 2002. Utilization of different nitrogen sources by marine cyanobacteria Prochlorococcus and Synechococcus. Limnol. Oceanogr. 47:989-996.
  18. Partensky, F., J. Blanchot, and D. Vaulot. 1999. Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters: a review. Bull. Inst. Oceanogr. 19:457-476.
  19. Penno, S., D. Lindell, and A. F. Post. 2006. Diversity of Synechococcus and Prochlorococcus populations determined from DNA sequences of the N-regulatory gene ntcA. Environ. Microbiol. 8:1200-1211.[CrossRef][Medline]
  20. Post, A. F. 2005. Nutrient limitation of marine cyanobacteria: molecular ecology in the oligotrophic sea, p. 87-107. In J. Huisman, H. C. P. Matthijs, and P. M. Visser (ed.), Harmful cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  21. Sakamoto, T., K. Inoue-Sakamoto, and D. A. Bryant. 1999. A novel nitrate/nitrite permease in the marine cyanobacterium Synechococcus sp. strain PCC 7002. J. Bacteriol. 181:7363-7372.[Abstract/Free Full Text]
  22. Scanlan, D. J. 2003. Physiological diversity and niche adaptation in marine Synechococcus. Adv. Microb. Physiol. 47:1-64.[Medline]
  23. Strickland, J. D. H., and T. R. Parsons. 1972. A practical handbook of seawater analysis. Fisheries Research Board of Canada bulletin, 2nd ed. Fisheries Research Board of Canada, Ottawa, Ontario, Canada.
  24. Suzuki, I., N. Horie, T. Sugiyama, and T. Omata. 1995. Identification and characterization of two nitrogen-regulated genes of the cyanobacterium Synechococcus sp. strain PCC 7942 required for maximum efficiency of nitrogen assimilation. J. Bacteriol. 177:290-296.[Abstract/Free Full Text]
  25. Ting, C. S., G. Rocap, J. King, and S. W. Chisolm. 2002. Cyanobacterial photosynthesis in the oceans: the origins and significance of divergent light-harvesting strategies. Trends Microbiol. 10:134-142.[CrossRef][Medline]
  26. Vázquez-Bermúdez, M. F., J. Paz-Yepes, A. Herrero, and E. Flores. 2002. The NtcA-activated amt1 gene encodes a permease required for uptake of low concentrations of ammonium in the cyanobacterium Synechococcus sp. PCC 7942. Microbiology 148:861-869.[Abstract/Free Full Text]
  27. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can. Bull. Fish Aquat. Sci. 214:71-120.
  28. Wyman, M., R. P. F. Gregory, and N. G. Carr. 1985. A novel role for phycoerythrin in a marine cyanobacterium Synechococcus strain DC2. Science 230:818-820.[Abstract/Free Full Text]
  29. Zubkov, M. V., B. M. Fuchs, G. A. Tarran, P. H. Burkill, and R. Amman. 2003. High rate of uptake of organic nitrogen compounds by Prochlorococcus cyanobacteria as a key to their dominance in oligotrophic oceanic waters. Appl. Environ. Microbiol. 69:1299-1304.[Abstract/Free Full Text]

Applied and Environmental Microbiology, May 2007, p. 3028-3033, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02606-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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