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

Constructs.To integrate a fusion of the Synechococcus sp. strain PCC7942 GS-encoding gene (glnA) promoter with V. harveyi luxAB into the cyanobacterial chromosome, a plasmid (pGSL) was constructed. The plasmid was based on plasmid pAM1414 (2), which contains the luxAB luciferase genes (V. harveyi) and a spectinomycin-streptomycin resistance cartridge. It also harbors the intergenic regions of a neutral site (NSI; GenBank accession number U30252 ) of Synechococcus sp. strain PCC7942 chromosomal DNA which allow for the integration of the plasmid into the chromosome by double homologous recombination (29). A 413-bp fragment that included the glnA promoter (GenBank accession number D26162 ) was cloned by PCR with a 24-base primer (5′-GATTAAGCGGCCGCTCCCGAGTG-3′) at the 5′ end of the promoter and a 25-base primer (5′-CATTAAGGATCCAGGCCTGAGCGAC-3′) at its 3′ end. The 410-bp promoter segment was inserted into the NotI/BamHI site of the plasmid, upstream of luxAB. The amplified segment included 9 bp upstream of the start codon of glnA, which was eliminated (T was replaced by G). The location and orientation of the fusion were confirmed by PCR and by restriction analysis with PvuII.

Wild-type Synechococcus sp. strain PCC7942 was transformed with recombinant and control plasmids pGSL and pAM1414 as previously described (19) to generate strains GSL (PglnA::luxAB) and M1414 (control), respectively. Transformed cells were selected on agar plates that contained streptomycin and spectinomycin (25 μg ml⁻¹ each) in BG11 medium (39). E. coli DH5α, used for plasmid propagation, was grown on Luria-Bertani medium (33) supplemented with spectinomycin and/or streptomycin (50 μg ml⁻¹ each) when appropriate.

Southern analysis.Southern blot hybridization, performed as described previously (18), was used to confirm the integration of pGSL into the target site (NSI) in the Synechococcus sp. strain PCC7942 chromosome.

Growth and experimental conditions.Wild-type Synechococcus sp. strain PCC7942 and its derived PglnA::luxAB engineered strain (GSL) were grown as batch cultures at 30°C in BG11 mineral medium (39) under constant fluorescent illumination (50 μmol of photons m⁻² s⁻¹) with continuous shaking (Innova 4340; New Brunswick Scientific Co., Edison, N.J.; 125 rpm). For N deprivation experiments, the cells were pregrown to an optical density at 750 nm (OD₇₅₀) of approximately 0.7. Cells were then harvested by centrifugation (4,000 rpm in Eppendorf centrifuge 5810R for 10 min at room temperature), washed twice in N-free BG11 medium (NaNO₃ and ferric ammonium sulfate were replaced by equimolar concentrations of NaCl and ferric chloride, respectively) without antibiotics, and resuspended in the same medium to a final OD₇₅₀ of 0.25.

For experiments with different N sources, cultures were harvested as described above and resuspended in N-free BG11 medium containing ammonium (as NH₄Cl), nitrate (NaNO₃), nitrite (NaNO₂), l-glutamine, or urea at various concentrations. All N sources used in this study were obtained from Sigma Chemical Co.

For experiments involving the readdition of N to N-deprived cells, the cells were first incubated for 10 h in N-free BG11 medium as described above and then supplemented with various ammonium concentrations.

Determination of ammonium concentrations.Ammonium concentrations were determined by using a QuikeChem 8000 flow injection analyzer (Lachat Instruments).

Measurement of bioluminescence.The bioluminescence of the cyanobacterial constructs was assayed as described previously (18). Cell aliquots were brought to a uniform cell density (OD₇₅₀, 0.5), and duplicate subsamples (100 μl) of the cell suspension were transferred to wells of an opaque white 96-well microtiter plate (Nunc, Roskilde, Denmark). The reaction was started by the addition of 100 μl of N-free BG11 medium containing the luciferase substrate nonyl aldehyde (0.002%) and 0.005% Igepal CA-630. Luminescence was measured with a temperature-controlled (30°C) microtiter plate luminometer (Victor²; Wallac, Turku, Finland) every 10 s for 15 min. Light emission increased to a maximum during the first 5 min and then declined. The maximum luminescence at the peak is the value used in this communication. Light output intensity is reported either in the instrument's arbitrary relative light units (RLU) or as a percentage of the activity of the fully induced system in N-free medium. All experiments were conducted in duplicate and were repeated at least twice.

Measurement of GS activity.GS transferase activity was determined over 15 min at 37°C as previously described (48). The assay quantifies the GS-mediated transformation of glutamine into γ-glutamylhydroxamate (γ-GH), the concentration of which is determined photometrically. Culture aliquots (5 to 10 ml) were harvested by centrifugation (4,000 rpm in Eppendorf centrifuge 5810R for 10 min at room temperature) and resuspended in 0.5 mM Tris HCl (pH 7.5). Toluene was added (one-half of the cell suspension volume), and the mixture was vortexed vigorously for 60 s. Cells were then washed three times with the Tris HCl solution by centrifugation (14,000 rpm in Eppendorf centrifuge 5417C for 2 min at 4°C) and resuspended in a reaction mixture containing 135 mM imidazole HCl, 18 mM hydroxylamine HCl, 0.27 mM MnCl₂, 25 mM potassium arsenate, 0.36 mM sodium ADP, and 1 mg of hexadecyltrimethylammonium bromide/ml. The pH of the reaction mixture was adjusted to 7.55 with 2 M KOH, and the cell mixture was equilibrated for 15 min at 37°C. l-Glutamine (0.015 mM) was then added to the mixture, which was incubated for an additional 15 min at the same temperature. A “stop mixture” (1 ml) containing 0.2 M FeCl₃ · 6H₂O, 0.122 M trichloroacetic acid, and 0.175 M HCl was used to terminate the enzymatic reaction, and the samples were centrifuged (4 min at 7,000 rpm in Eppendorf centrifuge 5417C) to remove the cells. The OD₅₄₀ of the supernatant was measured, and γ-GH concentrations were calculated by using a standard curve. Protein in the cell extracts was determined by the method of Bradford (5) with bovine serum albumin as a standard. GS activity values are presented as micromoles of γ-GH produced per hour per milligram of protein or as a percentage of maximal GS activity determined with N-deprived cells at the same time point. All chemicals used in this assay were obtained from Sigma.

Field samples.Water samples were collected from Lake Kinneret, Israel, a warm monomictic freshwater lake with mean and maximum depths of 24 and 42 m, respectively. Monthly water samples were collected from November 2000 until December 2001 at the deepest point near the center of the lake (station A). The sampling depths chosen spanned both the epilimnion (1, 5, and 10 m) and the hypolimnion (30 and 37 m). To prevent biological activity that might have affected nutrient concentrations, the water samples were filter sterilized (0.22-μm-pore-size filter; Schleicher & Schuell) within 2 h of sampling and kept at 4°C until analyzed.

Total dissolved N (TDN) in Lake Kinneret water samples was calculated as the sum of dissolved (0.45-μm-pore-size filter) ammonium, nitrate, nitrite, and organic N; each type of N was assayed by standard methods (1), with the exception of the use of Szechrome NAS reagent (diphenylamine sulfonic acid chromogen) (45) for NO₃⁻ assays.

Assays of N bioavailability in lake water samples.Assaying for bioavailable N with reporter strain GSL developed in this study was performed as described previously for phosphorus bioavailability (18). Within 24 h of collection, duplicate samples (10 ml) were transferred to 50-ml sterile flasks and supplemented with N-free BG11 medium ingredients. N-replete GSL cells, pregrown and washed as described above (growth and experimental conditions), were then added to a final OD₇₅₀ of approximately 0.25. Cells suspended in complete or N-free BG11 medium served as negative or positive controls, respectively. Samples were incubated at 30°C with continuous shaking (125 rpm) under constant fluorescent illumination (50 μmol of photons m⁻² s⁻¹). Following 20 h of incubation, bioluminescence was assayed by using opaque white 96-well microtiter plates as described above.

Verification of strain GSL chromosomal integration.A PglnA::luxAB fusion was introduced into pAM1414 (2) to yield plasmid pGSL, which was then incorporated into a neutral cloning site (NSI) on the Synechococcus sp. strain PCC7942 chromosome (7). Homologous recombination at the target site was verified by Southern analysis with a fluorescein-labeled NSI PCR product as a probe. ApaI and MfeI digests from the wild-type strain and strain GSL were hybridized with the probe (Fig. 1). In strain GSL, the NSI probe hybridized to a larger fragment. The difference in size (approximately 5,000 bp) corresponded to the size of the insert within the NSI in plasmid pGSL (4,894 bp), confirming that it was introduced into the genome at the NSI by a double-recombination event. The location and orientation of the insert were further confirmed by PCR (data not shown).

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FIG. 1.

Southern analysis of ApaI and MfeI digests of genomic DNAs of wild-type Synechococcus sp. strain PCC7942 and strain GSL. The blot was hybridized with a fluorescein-labeled probe based on a 382-bp NSI PCR product. Numbers on the right indicate molecular weights.

Response of Synechococcus sp. strain GSL to N availability.Upon transfer to an N-free medium, both wild-type and GSL (harboring the PglnA::luxAB fusion) Synechococcus cultures underwent one slow cell division within 8 to 12 h and then ceased to grow. The N-deprived cultures turned from green to yellowish green approximately 12 h after N depletion as previously described (11, 13). In spite of the obvious stress exhibited under these conditions, the cells responded to N deprivation by luciferase synthesis, as detected from the emission of luminescence within 2 to 5 h after transfer. Luminescence continued for at least 20 h (Fig. 2A). When the cells were transferred to an N-replete medium (1 mM ammonium), no luminescence above the background level (approximately 10% the maximum luminescence measured) was observed. Lower ammonium concentrations led to a progressively higher degree of bioluminescence (Fig. 2A). The N dose dependence is shown in Fig. 2B, in which luminescence at 20 h is plotted as a function of ammonium concentration. Strain GSL appeared to be responsive to ammonium concentrations of between 1 mM and 1 μM. Figure 2B also shows the GS activities of the same cultures at the same time point, indicating that the activity of this enzyme was a far less sensitive indicator of N availability: activities were similar at all tested concentrations below 1 mM. No luminescence was observed for Synechococcus sp. strain M1414 control cells, which lacked the glnA promoter upstream of the luxAB genes. Neither did phosphorus limitation induce luminescence in strain GSL (data not shown). In all cases, the inorganic carbon (240 μM) present in BG11 mineral medium ensured that GS induction and activity were not affected by carbon limitation.

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FIG. 2.

Bioluminescence in Synechococcus sp. strain GSL. (A) Luminescence development in the presence of different NH₄⁺ concentrations. (B) Light emission and GS activity as a function of NH₄⁺ concentration following 20 h of exposure. Data in panel B (averages and standard deviations) represent the percent maximal luminescence or GS activity obtained at that time point in N-free medium in three replicate experiments (917.9 ± 120.4 RLU or 443.4 ± 34.0 μmol of γ-GH h⁻¹ mg of protein⁻¹, respectively).

To better characterize the response of strain GSL to ammonium, the cells were exposed to a 100 μM concentration of this ion; luminescence, GS activities, and ammonium concentrations were monitored at short time intervals (Fig. 3). Within approximately 5 h, the ammonium concentration decreased to ca. 10 μM, at which time both luminescence and GS activity started to increase. These results indicate that during the increasingly longer lag periods observed earlier (Fig. 2A) in the presence of higher ammonium concentrations, free ammonium was first consumed and only then was PglnA-driven bioluminescence induced. The ammonium concentration below which induction occurred under these conditions was about 10 μM.

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FIG. 3.

NH₄⁺ uptake, bioluminescence, and GS activity induction in Synechococcus sp. strain GSL. Mid-log-phase cells grown in complete BG11 medium were transferred at time zero to N-free medium containing 100 μM NH₄⁺. The remaining NH₄⁺, bioluminescence, and GS activity were determined at intervals.

Synechococcus sp. strain PCC7942, like other cyanobacteria, can utilize diverse N sources in addition to ammonium. These include nitrate, nitrite, and various organic N compounds (39). Figure 4 summarizes the bioluminescence and GS activity of strain GSL cells 20 h after exposure to four different N sources at various concentrations: nitrate (Fig. 4A), nitrite (Fig. 4B), glutamine (Fig. 4C), and urea (Fig. 4D). In all cases, a clear dependence of bioluminescence intensity on the initial N concentration was observed; the detection range appeared to be broader for the inorganic N sources, extending from 1 mM to 1 μM. For glutamine and urea, the lowest concentrations detected in this manner were about 10 and 50 μM, respectively. GS activity, as in the case of ammonium, was a far less sensitive indicator of N source concentration; in most cases, with the possible exception of urea, below a certain high concentration GS activities were uniformly high and thus could not serve as an indicator of N levels.

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FIG. 4.

Bioluminescence and GS activity in GSL cells 20 h after transfer to N-free BG11 medium supplemented with nitrate (A), nitrite (B), glutamine (C), or urea (D). Data (averages and standard deviations; three replicate experiments) represent the percent light output (▪) or GS activity (•) obtained at that time point in the maximally induced (N-free) control. Average bioluminescence and GS control (100%) values were 576.8 ± 60.0, 782.8 ± 166.6, 685.8 ± 235.7, and 1,056.8 ± 98.6 RLU and 325.4 ± 12.1, 403.2 ± 62.8, 330.3 ± 16.1, and 368.3 ± 32.4 μmol of γ-GH h⁻¹ mg of protein⁻¹ for nitrate, nitrite, urea, and glutamine, respectively.

The experiment shown in Fig. 5 was designed to characterize the response reciprocal to that shown in Fig. 2: N-depleted (and thus preinduced) cultures were confronted with a renewed NH₄⁺ supply. The cells were prestarved for N for 10 h and then transferred to media with different NH₄⁺ concentrations (Fig. 5A). In an N-free medium, luminescence continued to increase for approximately an additional 10 h, whereas at an NH₄⁺ concentration of 1 mM, light emission was completely inhibited within the same time period. For intermediate concentrations, a temporary decrease in luminescence was followed by a renewed increase after more than 5 h (0.1 and 0.01 mM) or 15 h (0.5 mM). The decline, representing a temporary cessation of glnA promoter activation and a lack of luciferase protein synthesis, probably lasted for as long as NH₄⁺ was available. The observed luminescence at each time point reflected the balance between the deterioration of “old” luciferase activity from the preinduction period and the synthesis of “new” enzyme in response to renewed N depletion. GS activity, monitored in parallel in the same samples, also responded to the renewed presence of ammonium, but in a significantly less sensitive manner. This difference between the two activities is demonstrated in Fig. 5B, which shows luminescence and GS activity 10 h after ammonium readdition. From the kinetic data in Fig. 5A, luciferase and GS half-lives of approximately 3 to 4 h and 10 h, respectively, were calculated.

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FIG. 5.

Effects of the addition of NH₄⁺ to preinduced (10 h in N-free medium) GSL cells. (A) Luminescence development in the presence of different NH₄⁺ concentrations. (B) Light emission (▪) and GS activity (•) as a function of NH₄⁺ concentration following 10 h of exposure. Data in panel B (averages and standard deviations) represent the percent maximal luminescence or GS activity obtained at that time point in N-free medium in three replicate experiments (661.7 ± 117.4 RLU and 360.7 ± 77.3 μmol of γ-GH h⁻¹ mg of protein⁻¹, respectively).

N bioavailability in Lake Kinneret (Sea of Galilee).To test the applicability of the developed method, water samples were collected monthly from Lake Kinneret, Israel's largest lake and main water reservoir. The lake, 42 m at its deepest, is isothermal during the winter and stratified in spring, summer, and fall, with a thermocline at a depth of 18 to 25 m (22). Over a 12-month period, a profile was sampled monthly along the upper (1, 5, and 10 m) and lower (30 and 37 m) layers of the lake.

N-replete GSL cells were exposed to these samples, and luminescence was recorded 20 h later. Representative examples of two such profiles are shown in Fig. 6A and C, along with the total dissolved N (TDN) measured chemically in the same water samples. Also shown are the temperature profiles (Fig. 6B and D) in the lake at the time of sampling. N bioavailability, as indicated by the luminescence pattern in both sampling periods, clearly mirrored both the stratification (as evidenced by the temperature profile) and the TDN concentrations. The latter parameter encompasses ammonium, nitrate, nitrite, and organic N sources; none of these individual N compounds displayed the same degree of correlation to the bioluminescence profiles as TDN (data not shown). This result is understandable in view of the fact that strain GSL responds to a different degree to each of the potential N sources; since all of them were represented to some extent in the water samples, only a global parameter may reflect the total potentially available N.

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FIG. 6.

Bioluminescence in Synechococcus sp. strain GSL in water samples collected from Lake Kinneret in February 2001(A and B) and September 2001 (C and D). Bioluminescence (▪) measured 20 h after inoculation is plotted along with TDN (▴) and temperature (•, ⧫). The temperature data (B and D) demonstrate the stratification (September) and mixing (February) of the lake at the time of sampling.

To present a more comprehensive profile of N availability in Lake Kinneret throughout the year, bioluminescence and TDN results for all upper-layer (1, 5, and 10 m) and lower-layer (30 and 37 m) samples were pooled, averaged, and plotted as a function of the sampling month (Fig. 7). Also depicted in Fig. 7 are the average chlorophyll a concentrations for the same water samples, representing the density of the phytoplankton populations. In the upper layer (Fig. 7A), TDN concentrations were comparatively low (0.36 to 0.73 mg liter⁻¹) throughout the year, yielding correspondingly high bioluminescence. In the lower layer (Fig. 7B), N concentrations were high during stratification (March to December) and low during the mixed period (January and February), changes that were consistently reflected by the bioluminescence of strain GSL in the same samples. The seasonal chlorophyll development in the lake followed a clear monomictic pattern with a single annual peak: concentrations were low for most of the year, with a single significant increase during the mixed period in January and February. The mixing effect led to a substantial decrease in TDN concentrations in the hypolimnion but only to a moderate increase in the epilimnion. Both of these effects were clearly mirrored by strain GSL bioluminescence.

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FIG. 7.

Light output of GSL cells measured 20 h after inoculation and chlorophyll and TDN concentrations in monthly epilimnion (A) (average of 1-, 5-, and 10-m samples) and hypolimnion (B) (average of 30- and 37-m samples) Lake Kinneret samples. Data are averages and standard deviations.

While the correlation between TDN and bioluminescence was obvious, it was not sufficient for assessing the degree of availability of the chemically determined nutrient. For this purpose, we used the calibration curve plotted in Fig. 2B to calculate the concentration of “available N” indicated by the measured bioluminescence of strain GSL. We carried out this calibration by using the most available N species, ammonium. The equation used was based upon the data in Fig. 2B: y =−10.89ln(x) + 46.155 (R² = 0.96); in this equation, y corresponds to light output (as a percentage of the maximum luminescence obtained in N-free medium) and x corresponds to initial NH₄⁺ concentration (in milligrams liter⁻¹). Because ammonium was used as the reference, our availability results may be considered in terms of “ammonium equivalents.” The luminescence data in Fig. 7 were transformed in this manner and are plotted in Fig. 8 against chemically determined TDN concentrations in the same samples. The emerging general pattern is clear: the lower the overall N concentration, the lower the available fraction out of this total. Thus, in the lower layer, characterized by high N concentrations, calculated availability values were similar to or even higher than measured ones, indicating that under these conditions, practically all of the nutrient was in a form available to the reporter strain. TDN concentrations were up to 10-fold lower in the epilimnion than in the hypolimnion. This depletion, probably caused by uptake of the more available N species, led to an increase in the fraction of less available N. Even under these conditions, however, N availability was rarely estimated to be lower than 5% of the TDN, or 0.04 mg liter⁻¹.

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FIG. 8.

Available N concentrations, calculated as ammonium equivalents and plotted as a function of TDN in the same samples. Symbols: •, epilimnion samples; ○, hypolimnion samples.