Mutual Dependence of Nitrogen and Phosphorus as Key Nutrient

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Environmental Processes

Mutual dependence of nitrogen and phosphorus as key nutrient elements: one facilitates Dolichospermum flos-aquae to overcome limitation by the other Siyang Wang, Jian Xiao, Lingling Wan, Zijun Zhou, Zhicong Wang, Chunlei Song, Yiyong ZHOU, and Xiuyun Cao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04992 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Environmental Science & Technology

Abstract Graphic 425x239mm (300 x 300 DPI)

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ABSTRACT: Dolichospermum flos-aquae (formerly Anabaena flos-aquae) is a

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diazotrophic cyanobacterium causing harmful blooms worldwide, which is partly

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attributed to its capacity to compete for nitrogen (N) and phosphorus (P). Preventing

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the blooms by reducing P alone or both N and P has caused debate. To test the effects

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alone and together on the growth of cyanobacteria, we performed culture experiments

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in different eutrophication scenarios. N2 fixation in terms of heterocyst density,

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nitrogenase activity and nifH expression increased significantly in P-replete cultures,

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suggesting that P enrichment facilitates N2 fixation. Correspondingly, the expression

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of genes involved in P uptake, e.g., those involved in P-transport (pstS) and the

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hydrolysis of phosphomonoesters (phoD), was upregulated in P-deficient cultures.

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Interestingly, N addition enhanced not only the expression of these genes but also

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polyphosphate formation and alkaline phosphatase activity in P-deficient cultures

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relative to the P-replete cultures, as evidenced by qualitative (enzyme-labeled

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fluorescence) and quantitative (fluorogenic spectrophotometry) measurements.

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Furthermore, after N addition, cell activity and growth increased in the P-deficient

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cultures, underscoring the risk of N enrichment in P-limited systems. The

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eco-physiological responses shown here help further our understanding of the

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mechanism of N and P colimitation and underscore the importance of dual N and P

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reduction in controlling cyanobacterial blooms.

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Key words: N 2 -fixing cyanobacteria; phosphorus; nitrogen; mutual dependence;

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colimitation; gene expression

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INTRODUCTION Eutrophication and cyanobacterial blooms pose risks to human and ecosystem

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health and have been attributed to over-enrichment with phosphorus (P) and nitrogen

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(N) in water bodies.1 Dual N and P reduction has become central to water

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management because N and P colimitation of primary productivity has been shown to

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be a potentially important process in freshwater environments.2 However, an

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argument against combined N and P control is that reducing only P can successfully

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curb lake eutrophication, while it cannot be controlled by reducing N input because N2

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fixation can alleviate N deficits, making N control irrelevant to eutrophication

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management.3−5 On the other hand, this behavior has been shown not to be true for

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some eutrophic systems where N was also important for the control of

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eutrophication.6−8

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The biochemical coupling of N and P on the cellular level remains unclear. Liebig's

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Law of the Minimum has been used to argue against nutrient colimitation of growth.9

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However, in many cases, N and P have been shown to affect the growth rate

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simultaneously rather than sequentially.10−11 Colimitation of N and P is biochemically

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independent.12 In contrast, others thought that N and P were biochemically dependent,

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assuming that P quota enhances N uptake.11 An investigation of the combined effects

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of N and P on phytoplankton growth at physiological and ecological levels would

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clarify this issue.

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The growth of Dolichospermum spp. (formerly Anabaena spp.), bloom-forming, cyanotoxin-producing cyanobacteria, is more likely to be limited by P rather than N



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since they can fix atmospheric N2 into biologically available NH3.3,13 Furthermore, N2

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fixation, a metabolically expensive process, is controlled by P availability.6

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Cyanobacteria have developed several strategies in response to low inorganic P (Pi)

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supply, thereby, reducing their P-requirements,14 using cellular phosphate and/or

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enhancing Pi acquisition by synthesizing phosphate transporters15,16 and increasing the

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affinity and rate of Pi uptake.17 The P-specific transport (Pst) system comprises a

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periplasmic Pi-binding protein (PstS) and sensitively responds to P deficiency.18 The

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ability to use the dissolved organic phosphorus (DOP) pool, mediated by alkaline

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phosphatase (APase), is an additional strategy for P acquisition. The three most

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important phosphatase genes (phoX, phoA and phoD) have been recognized within

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marine microorganisms.19 Protein structure analysis revealed that phoD encodes

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APase in Anabaena.20 Anabaena sp. always produces extracellular APase, as

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evidenced by enzyme-labeled fluorescence (ELF) in the natural waters.21 Another

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strategy used by microbes to survive Pi-deficiency is that they store Pi in the form of

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polyphosphate (poly-P) in cells when P is abundant, followed by the break-down

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poly-P stores upon P stress.22

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This study investigated whether N also plays a role in P scavenging as P does in N

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utilization. We measured the transcript levels of genes involved in Pi assimilation

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(pstS), DOP hydrolysis (phoD) and N2 fixation (nifH); the quantity and quality of

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APase and poly-P body (PPB) formation in monocultures of D. flos-aquae FACHB

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245 under N- or P-replete and N- or P-deficient conditions. These experiments

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provided insight into the molecular and eco-physiological responses of N 2 -fixing


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cyanobacteria to varying exogenous N and P concentrations to test the hypothesis that

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N and P metabolism is biochemically dependent in cyanobacteria and therefore

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underscores the need for dual N and P reduction in controlling eutrophication and

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cyanobacterial blooms. The laboratory-scale experiments help to develop an

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understanding of microbial nutrient cycling in natural environments.

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MATERIALS AND METHODS

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2.1. Experimental design

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D. flos-aquae FACHB 245 was obtained from the Freshwater Algae Culture

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Collection of Institute of Hydrobiology (FACHB-collection, Wuhan, China). Prior to

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experiments, D. flos-aquae cells were harvested in the logarithmic growth period by

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centrifugation (4,000 rpm, 8 min), rinsed 3 times with N- and P-free BG11 medium23

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and then inoculated in medium for 48 h to exhaust nutrients stored in cells. Two

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experiments probing the responses of gene expression and N2 fixation to varying

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concentrations of P and N were performed separately.

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2.1.1. Experiment on responses of gene expression to varying concentrations of P and

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N

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At the onset of the experiment, monocultures of D. flos-aquae were exposed to two P concentrations (-P, 0.05 mg L−1; +P, 2 mg L−1 , mimicking P-deficient and P-replete



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status, respectively) with 0.5 mg NO3−-N L−1 (mimicking N-deficient conditions) in

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triplicate. The different nutrient treatments were obtained by modifying the nitrate

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(NaNO3) and phosphate (KH2PO 4) concentrations in regular BG11 medium. On day 9,

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we added nitrate (NaNO3) to the treatments (final concentration, 2 mg L−1). The

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experiment was conducted in 500 mL Erlenmeyer flasks filled with 200 mL of

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medium containing D. flos-aquae and lasted 16 days. The experimental units were

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maintained in conditions suitable for cyanobacterial growth: constant photoperiod (12

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h of light: 12 h of dark), 30 μmol photons m−2 s−1 irradiance, and 25 °C. Subsamples

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were collected every two days for cell density, chemical, and enzymatic analysis and

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quantitative real-time PCR (qPCR) to estimate the expression of phoD, pstS and nifH

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genes.

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2.1.2. Determining responses of N2-fixation to varying concentrations of P and N Monocultures of D. flos-aquae were exposed to different P concentrations (0 mg

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L−1, 0.05 mg L−1 , 0.5 mg L−1, and 2 mg L−1) at 0.5 mg NO3−-N L−1 (mimicking

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N-deficient conditions) and different N concentrations (0 mg L−1 , 0.5 mg L −1, 2 mg

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L−1, and 10 mg L−1) at 0.05 mg L−1 P in triplicate. The experiment lasted 8 days.

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Subsamples were examined for heterocyst density and nitrogenase activity every two

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days.

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2.2. Biological, chemical and biochemical analysis

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Samples for cell and heterocyst density estimation were preserved with Lugol’s

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solution and counted with an Olympus BX 41 microscope (Olympus Corporation,

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Japan). Chlorophyll fluorescence was examined by Phyto-PAM (Walz, Effeltrich), the

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photochemical efficiency of photosystem II (Fv /Fm) was determined according to Ting

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& Owens,24 and the values of maximum relative electron transport rate (rETRmax)

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were plotted vs irradiance to give the corresponding rapid light curve, which was

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automatically recorded by preset software in the Phyto-PAM.25

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Samples were processed by filtering through 0.45 μm (Millipore) filters, and an

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analysis of soluble reactive phosphorus (SRP) and nitrate (NO3 −-N) concentrations by

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a standard method26,27 was then performed. Alkaline phosphatase activity (APA) was

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quantified by fluorometric assay.28 Nitrogenase activity was analyzed by the acetylene

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reduction assay.29 Qualitative levels of extracellular APases from D. flos-aquae were

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detected using ELF 97 phosphate (Molecular Probes) on the basis of the protocol

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proposed by Štrojsová et al.21

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PPBs are linear polymers of several to several hundred phosphate groups and are

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found extensively in microbial cells;30 many microorganisms and even cyanobacteria

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can excessively absorb Pi and store it in cells in the form of PPBs.22 Fluorescence

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staining by 4’,6-diamidino-2-phenylindole (DAPI) was used to examine D. flos-aquae

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for PPBs with epifluorescence microscopy (Olympus BX51FL) with UV excitation

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according to the procedure of Bar-Yosef et al.31

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2.3. Molecular analysis



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D. flos-aquae cells from culture samples were concentrated and pelleted by

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centrifugation.32 Total RNA was extracted for analysis by using the Bioline ISOLATE

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II RNA Mini kit, following the manufacturer’s protocol, and we used DNase MaxTM

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Kits (MoBio, USA) to remove the total DNA. The purity and yield of RNA were

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assessed with a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific, USA).

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cDNA was reverse transcribed from RNA using the Promega GoScriptTM Reverse

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Transcription System.

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The primers for pstS, phoD and rpoC1 (housekeeping gene) amplification were

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identified from CyanoBase (http://genome.microbedb.jp/cyanobase) and designed

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using Primer-BLAST (Supplemental Methods, Table 1). Moreover, we ran BLASTN

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to compare primers against other genomes to ensure their validity and specificity.

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Triplicate reactions were carried out with SYBR® Green PCR Master Mix, and the

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reaction system (20 μL) contained 10 μL of 2 × Mix, 0.4 μM forward and reverse

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primers and 1 μL of 1:10 diluted cDNA. qPCR was conducted in an Applied

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Biosystems 7900 Real-Time PCR System (Applied Biosystems, USA), and the qPCR

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program for target genes was 94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s,

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53 °C for 30 s, and 72 °C for 30 s, and followed by 1 step of 72 °C for 7 min. To

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normalize the qPCR data, we chose the RNA polymerase gene rpoC1 as a suitable

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housekeeping gene for the analysis of gene expression levels. Fold changes in gene

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transcript levels were calculated as 2 −

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amplification efficiency values of target genes and housekeeping gene must be

△△Ct

. For the 2

−△△Ct


equation to be valid, the

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approximately equal (less than 0.1). We estimated the transcript abundances of phoD,

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pstS, and nifH within cDNA samples based on the known copy numbers of linearized

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plasmids of phoD, pstS, and nifH gene clones. Finally, the values were calculated as

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gene transcript abundances (copies mL−1 culture). The amplification efficiencies of

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primer pairs were 97.8–105.5% (R2 >0.99) (Table 1).

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2.4. Statistical analysis Both two-tailed Pearson’s correlation tests and analyses of variance were performed

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with SPSS 18.0 (Chicago, USA) to examine the relationships or differences between

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parameters, and differences were considered statistically significant at P< 0.05 or P

Mutual Dependence of Nitrogen and Phosphorus as Key Nutrient

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