Acclimation of Nodularia spumigena CCY9414 to inorganic phosphate limitation - Identification of the P-limitation stimulon via RNA-seq

Abstract

Nodularia spumigena is a toxic, filamentous cyanobacterium capable of fixing atmospheric N₂, which is often dominating cyanobacterial bloom events in the Baltic Sea and other brackish water systems worldwide. Increasing phosphate limitation has been considered as one environmental factor promoting cyanobacterial mass developments. In the present study, we analyzed the response of N. spumigena strain CCY9414 toward strong phosphate limitation. Growth of the strain was diminished under P-deplete conditions; however, filaments contained more polyphosphate under P-deplete compared to P-replete conditions. Using RNA-seq, gene expression was compared in N. spumigena CCY9414 after 7 and 14 days in P-deplete and P-replete conditions, respectively. After 7 days, 112 genes were significantly up-regulated in P-deplete filaments, among them was a high proportion of genes encoding proteins related to P-homeostasis such as transport systems for different P species. Many of these genes became also up-regulated after 14 days compared to 7 days in filaments grown under P-replete conditions, which was consistent with the almost complete consumption of dissolved P in these cultures after 14 days. In addition to genes directly related to P starvation, genes encoding proteins for bioactive compound synthesis, gas vesicles formation, or sugar catabolism were stimulated under P-deplete conditions. Collectively, our data describe an experimentally validated P-stimulon in N. spumigena CCY9414 and provide the indication that severe P limitation could indeed support bloom formation by this filamentous strain.

Keywords: alkaline phosphatase; cyanobacterial bloom; diazotroph; polyphosphate; toxin; transcriptomics; transport.

FIGURE 1

Physiological and biochemical assessment of Nodularia spumigena CCY9414 under P-replete (+P) and P-deplete (–P) conditions. Growth expressed as increase in dry weight (A). O-phosphate (PO₄^3–) concentration in the cultivation media during the second experiment iteration (B). Accumulation of polyphosphates in cells of N. spumigena (C). Bars represent means of individual measurements shown as single points. Asterisks indicate significant differences between the P-replete and the P-deplete cultures at each sampling time point. Significant differences between sampling time points are indicated by different lower-case (P-deplete) and upper-case (P-replete) letters. The results of the statistical tests can be found in the Supplementary Tables 1, 2.

FIGURE 2

Venn diagrams for the global quantitative comparison of expression changes in Nodularia spumigena CCY9414 under P-replete (+P) or P-deplete (–P) conditions. Number of up-regulated (A) and down-regulated (B) genes under P starvation in the comparison of P-deplete and P-replete conditions at day 7, P-deplete and P-replete conditions at day 14, and between P-replete conditions at day 14 and day 7, respectively. The RNA-seq experiment was conducted with samples from the second experiment iteration.

FIGURE 3

Assignment of differentially expressed genes to KEGG pathways. Up- and down-regulated genes are shown for the comparison of P-deplete and P-replete conditions at d7, P-deplete and P-replete conditions at d14, and between P-replete conditions at d14 and d7. (A) Number of differentially expressed genes per KEGG pathway. Asterisks indicate significant functional enrichment (X² goodness-of-fit test, p < 0.05, parentheses indicate expected frequencies below 1). Number of genes per pathway in the genome is given after the pathway name. (B) Number of pathways containing differentially expressed genes. The RNA-seq experiment was conducted with samples from the second experiment iteration.

FIGURE 4

Gene expression of phosphate-regulated gene cluster nsp7450 to nsp7590 in N. spumigena CCY9414 under P-replete (+P) and P-deplete (–P) conditions. Shaded bands show minimum and maximum gene expression (normalized as square-root transformed per base pair coverage per Gbp sequencing effort) at each experimental condition. Arrows indicate gene arrangement and orientation and were colored according to operon assignment as provided in Table 1. Locus tags and gene names are according to Table 1 (hypo: hypothetical protein).