A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.     

Phosphorylation of the PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity.

The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular state of nitrogen assimilation relative to CO2 fixation by being phosphorylated at a seryl residue. In this study, we first determined the location of the phosphorylated seryl residue within the PII amino acid sequence. The phosphorylation site exhibits an RXS motif, a recognition sequence characteristic for cyclic AMP-dependent protein serine kinases from eukaryotes. We established an in vitro PII phosphorylation assay to further analyze the PII kinase activity in Synechococcus sp. strain PCC 7942. ATP was used specifically as a phosphoryl donor, and the PII kinase activity was shown to be stimulated by alpha-ketoglutarate. Unlike the PII-modifying uridylyltransferase- and uridylyl-removing enzyme characterized in proteobacteria, the activity of the PII kinase from the cyanobacterium did not respond to glutamine.     

Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp: characterization,phylogeny and response to nutrient limitation

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202-2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high-light adapted) and SS120 (low-light adapted). We obtained approximately 100-fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of approximately 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55 degrees C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.     

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.     

Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942.

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.     

A simple method to preserve oceanic phytoplankton for flow cytometric analyses

A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small-sized picoplankton (below 2 microns) such as Synechococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger-sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.     

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.     

Distribution of Mycosporine-Like Amino Acids Along a Surface Water Meridional Transect of the Atlantic

The composition and abundance of mycosporine-like amino acids (MAAs) were investigated in the surface waters along a 13,000-km meridional transect (52° N to 45° S) in the Atlantic Ocean (Atlantic Meridional Transect programme: Cruise AMT 18: 4/10/2008-10/11/2008). MAAs were ubiquitous along the transect, although the composition of the MAAs was variable. Highest concentrations were in the far south (below 40° S; MAA >1 μg L(-1)) and in north subtropical equatorial region (NER: 0-25° N; MAA up to 0.8 μg L(-1)). Highest MAA relative to chlorophyll-a occurred in the NER (MAA/chl-a ratio between 2 and 5). MAA/chl-a significantly correlated with the preceding month's mean daily UV dose and with UV-B/UV-A. In the far south, high MAA concentrations coincided with high phytoplankton biomass, high nutrients and a deep mixed layer associated with the austral spring. Here, the phytoplankton community was dominated by micro- and nano-eukaryotes. At the NER, the high MAA/chl-a coincided with low nutrient concentrations, a shallow mixed layer depth (20-70 m) and to a lesser extent to a shallow nitracline (40-90 m). Here, the phytoplankton consisted primarily of picophytoplankton (0-0.2 μm), dominated by the pico-cyanobacteria Synechococcus sp. and Prochlorococcus sp. and by the nitrogen fixing filamentous cyanobacterium Trichodesmium. The low nitrate concentrations (<0.1 μmol L(-1)) at the NER suggest that nitrogen fixation was required for MAA production. Specific MAAs could not easily be assigned to particular groups of phytoplankton and we could not rule out the possibility that MAAs were associated with symbiotic cyanobacteria contained within heterotrophic dinoflagellates or diatoms.     

PHOSPHORUS BIOAVAILABILITY MONITORING BY A BIOLUMINESCENT CYANOBACTERIAL SENSOR STRAIN 1

Phosphorus (P) is widely considered to be the main nutrient limiting the productivity of freshwater phytoplankton, but an assessment of its bioavailability in natural samples is highly complex. In an attempt to provide a novel tool for this purpose, the promoter of the alkaline phosphatase gene, phoA, from Synechococcus sp. PCC 7942 was fused to the luxAB luciferase genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus sp. PCC 7942 genome to yield strain APL, which emitted light when inorganic P concentrations fell below 2.3 μM. Light emission of P‐deprived cells decreased rapidly upon inorganic P readdition. The reporter was demonstrated to be a sensitive tool for monitoring the bioavailability of both inorganic and organic P sources. In water samples taken from a natural freshwater environment (Lake Kinneret, Israel), the luminescence measured correlated with total dissolved phosphate concentrations.     

Uptake of 2-oxoglutarate in Synechococcus strains transformed with the Escherichia coli kgtP gene

A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-(14)C]oxoglutarate. Synechococcus sp. strain PCC 7942 was transformed with DNA constructs carrying the Escherichia coli kgtP gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The Synechococcus sp. strains bearing the kgtP gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-(14)C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of a Synechococcus sp. strain bearing the kgtP gene.     

Nutrient-phytoplankton relationships in a tropical meromictic soda lake

Seasonal variation through one year in total nitrogen (TN), total phosphorus (TP), phytoplankton biomass, phytoplankton species composition and other environmental factors were examined in Lake Sonachi, a tropical meromictic soda lake. Mean concentrations of TN and TP were 11 000 µg N l^-1 and 100 µg P l^-1, respectively. Maximum concentrations of TN and TP occurred in the monimolimnion. Phytoplankton biomass ranged from 350 to 1260 mg m^-3. Synechococcus bacillaris, a small coccoid cyanophyte, dominated the phytoplankton. The mean chlorophyll a concentration of 37 mg · m^-3 was a modest value when compared with those of other tropical soda lakes. High TN:TP ratios indicated phosphorus limitation in the lake.     

Variability in protist grazing and growth on different marine Synechococcus isolates

Grazing mortality of the marine phytoplankton Synechococcus is dominated by planktonic protists, yet rates of consumption and factors regulating grazer-Synechococcus interactions are poorly understood. One aspect of predator-prey interactions for which little is known are the mechanisms by which Synechococcus avoids or resists predation and, in turn, how this relates to the ability of Synechococcus to support growth of protist grazer populations. Grazing experiments conducted with the raptorial dinoflagellate Oxyrrhis marina and phylogenetically diverse Synechococcus isolates (strains WH8102, CC9605, CC9311, and CC9902) revealed marked differences in grazing rates-specifically that WH8102 was grazed at significantly lower rates than all other isolates. Additional experiments using the heterotrophic nanoflagellate Goniomonas pacifica and the filter-feeding tintinnid ciliate Eutintinnis sp. revealed that this pattern in grazing susceptibility among the isolates transcended feeding guilds and grazer taxon. Synechococcus cell size, elemental ratios, and motility were not able to explain differences in grazing rates, indicating that other features play a primary role in grazing resistance. Growth of heterotrophic protists was poorly coupled to prey ingestion and was influenced by the strain of Synechococcus being consumed. Although Synechococcus was generally a poor-quality food source, it tended to support higher growth and survival of G. pacifica and O. marina relative to Eutintinnis sp., indicating that suitability of Synechococcus varies among grazer taxa and may be a more suitable food source for the smaller protist grazers. This work has developed tractable model systems for further studies of grazer-Synechococcus interactions in marine microbial food webs.     

Luminescent whole-cell cyanobacterial bioreporter for measuring Fe availability in diverse marine environments

A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.     

DIFFERENCES IN GROWTH AND PHYSIOLOGY OF MARINE SYNECHOCOCCUS (CYANOBACTERIA) ON NITRATE VERSUS AMMONIUM ARE NOT DETERMINED SOLELY BY NITROGEN SOURCE REDOX STATE1

The preference of phytoplankton for ammonium over nitrate has traditionally been explained by the greater metabolic cost of reducing oxidized forms of nitrogen. This “metabolic cost hypothesis” implies that there should be a growth disadvantage on nitrate compared to ammonium or other forms of reduced nitrogen such as urea, especially when light limits growth, but in a variety of phytoplankton taxa, this predicted difference has not been observed. Our experiments with three strains of marine Synechococcus (WH7803, WH7805, and WH8112) did not reveal consistently faster growth (cell division) on ammonium or urea as compared to nitrate. Urease and glutamine synthetase (GS) activities varied with nitrogen source in a manner consistent with regulation by cellular nitrogen status via NtcA (rather than by external availability of nitrogen) in all three strains and indicated that each strain experienced some degree of nitrogen insufficiency during growth on nitrate. At light intensities that strongly limited growth, the composition (carbon, nitrogen, and pigment quotas) of WH7805 cells using nitrate was indistinguishable from that of cells using ammonium, but at saturating light intensities, cellular carbon, nitrogen, and pigment quotas were significantly lower in cells using nitrate than ammonium. These and similar results from other phytoplankton taxa suggest that a limitation in some step of nitrate uptake or assimilation, rather than the extra cost of reducing nitrate per se, may be the cause of differences in growth and physiology between cells using nitrate and ammonium.