Daily transcriptome changes reveal the role of nitrogen in controlling microcystin synthesis and nutrient transport in the toxic cyanobacterium, Microcystis aeruginosa

Harke and Gobler BMC Genomics (2015) 16:1068 DOI 10.1186/s12864-015-2275-9 RESEARCH ARTICLE Open Access Daily transcriptome changes reveal the role of nitrogen in controlling microcystin synthesis and nutrient transport in the toxic cyanobacterium, Microcystis aeruginosa Matthew J. Harke and Christopher J. Gobler* Abstract Background: While transcriptomics have become a valuable tool for linking physiology and ecology in aquatic microbes, the temporal dynamics of global transcriptomic patterns in Microcystis have rarely been assessed. Furthermore, while many microbial studies have explored expression of nutrient transporter genes, few studies have concurrently measured nutrient assimilation rates. Here, we considered how the global transcriptomic patterns and physiology of the cyanobacterium, Microcystis aeruginosa, changed daily as cells were grown from replete to deficient nitrogen (N) conditions and then back to replete conditions. Results: During N deprivation, Microcystis downregulated genes involved in photosynthesis and respiration, carbon acquisition, lipid metabolism, and amino acid biosynthesis while upregulating genes involved in N acquisition and transport. With increasing N stress, both the strength of expression and number of genes being differentially expressed increased, until N was restored at which point these patterns reversed. Uptake of 15N-labeled nitrate, ammonium and urea reflected differential expression of genes encoding transporters for these nutrients, with Microcystis appearing to preferentially increase transcription of ammonium and urea transporters and uptake of these compounds during N deprivation. Nitrate uptake and nitrate transporter expression were correlated for one set of transporters but not another, indicating these were high and low affinity nitrate transporters, respectively. Concentrations of microcystin per cell decreased during N deprivation and increased upon N restoration. However, the transcript abundance of genes involved in the synthesis of this compound was complex, as microcystin synthetase genes involved in peptide synthesis were downregulated under N deprivation while genes involved in tailoring and transport were upregulated, suggesting modification of the microcystin molecule under N stress as well as potential alternative functions for these genes and/or this toxin. Conclusions: Collectively, this study highlights the complex choreography of gene expression, cell physiology, and toxin synthesis that dynamic N levels can elicit in this ecologically important cyanobacterium. Differing expression patterns of genes within the microcystin synthetase operon in response to changing N levels revealed the potential limitations drawing conclusions based on only one gene in this operon. Keywords: Microcystis, Transcriptome, Microcystin, Nitrogen * Correspondence: School of Marine and Atmospheric Sciences, Stony Brook University, 239 Montauk Hwy, Southampton, NY 11968, USA © 2015 Harke and Gobler. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Harke and Gobler BMC Genomics (2015) 16:1068 Background The cyanobacterium Microcystis commonly forms blooms in temperate, freshwater ecosystems and can produce the hepatotoxin, microcystin. Toxic blooms of this species have led to beach closures, public water emergencies, and health concerns [1–3]. Anthropogenic nitrogen (N) loading can be an important factor in the occurrence of cyanobacteria blooms caused by non-diazotrophs such as Microcystis spp. [4–6]. For instance, field studies have demonstrated that N loading can promote blooms of Microcystis [7, 8] and toxic strains of Microcystis can outgrow non-toxic strains under high N levels [6, 9, 10]. There appears to be a link between N and microcystin production, whereby Ndeprived Microcystis cells display reduced transcription of microcystin synthetase genes and decreased microcystin content as compared to replete N cells [11]. This observation has been supported by culture studies [10, 12–15] and field studies that have found that increases in exogenous N concentrations have been associated with increases in microcystin in lakes [5, 11, 13–16]. While the role of phosphorus in controlling the growth and physiology of freshwater cyanobacteria has been well studied [17, 18], comparatively less is known regarding responses to dynamic levels of N. During nitrogen stress, some cyanobacteria are known to initially induce systems for uptake of multiple N-containing compounds [11, 19] and N acquired through these transporters is then converted to ammonium and assimilated through the glutamine sythetase-glutamate synthetase cycle (GS-GOGAT). When exogenous N availability becomes too low to supply internal N demands, cyanobacteria may begin to rely on internal stores of N to prolong growth and may downregulate photosynthesis [20–22]. Still, freshwater cyanobacterial studies exploring physiological linkages between gene responses to N limitation and changes in cell physiology have been limited, especially for toxic, freshwater genera such as Microcystis. Transcriptomics have become a valuable tool for investigating linkages between physiology and ecology in aquatic microbes. To date, most global transcriptomic studies have examined single time points or time series studies over a day/night cycle [11, 23–29] with the exception of limited microarray studies [22, 30]. In this study, we sought to document the daily global transcriptomic patterns of Microcystis aeruginosa over a one week period as cells were grown from replete to deficient N conditions and returned to replete conditions. To assess the choreography of gene expression and cell physiology we examined expression patterns of microcystin synthetase genes and genes relating to N transport, carbon acquisition, and photosynthesis in unison with levels of cellular microcystin and nutrients, as well as the uptake rate of nitrogenous compounds and bicarbonate. To our knowledge, the relationship between Page 2 of 18 the expression of transporter genes and the uptake of corresponding target compounds (as measured here via isotopic tracers) has never been documented in cyanobacteria. Methods Experimental design Experiments were conducted to track gene expression patterns of Microcystis aeruginosa as populations were grown from N-replete conditions into low N conditions and returned to replete conditions. Experiments were performed with Microcystis aerug (...truncated)