A mechanistic study of the influence of nitrogen and energy availability on the NH4+ sensitivity of nitrogen assimilation in Synechococcus

Abstract

In most algae, NO3- assimilation is tightly controlled and is often inhibited by the presence of NH4+. In the marine, non-colonial, non-diazotrophic cyanobacterium Synechococcus UTEX 2380, NO3- assimilation is sensitive to NH4+ only when N does not limit growth. We sequenced the genome of Synechococcus UTEX 2380, studied the genetic organization of the nitrate assimilation related (NAR) genes, and investigated expression and kinetics of the main NAR enzymes, under N or light limitation. We found that Synechococcus UTEX 2380 is a β-cyanobacterium with a full complement of N uptake and assimilation genes and NAR regulatory elements. The nitrate reductase of our strain showed biphasic kinetics, previously observed only in freshwater or soil diazotrophic Synechococcus strains. Nitrite reductase and glutamine synthetase showed little response to our growth treatments, and their activity was usually much higher than that of nitrate reductase. NH4+ insensitivity of NAR genes may be associated with the stimulation of the binding of the regulator NtcA to NAR gene promoters by the high 2-oxoglutarate concentrations produced under N limitation. NH4+ sensitivity in energy-limited cells fits with the fact that, under these conditions, the use of NH4+ rather than NO3- decreases N-assimilation cost, whereas it would exacerbate N shortage under N limitation.

Keywords: Ammonium; N metabolism; NtcA regulation; cyanobacteria; glutamine synthetase; limitation; nitrate reductase; nitrite reductase.

Fig. 1.

Phylogenetic tree based on the sequences of Rubisco large subunit.

Fig. 2.

Putative promoters of nitrate assimilation genes and their regulatory genes.

Fig. 3.

Nitrate reductase (NR) kinetics for Synechococcus UTEX 2380 cells cultured under either N (□) or energy (○) limitation. The NR activity is shown as a function of NO₃⁻ concentration on a per cell (A, C) and per mg of protein (B, D) basis. (C, D) enlarged views of the first phase of the curves in (A, B). The error bars indicate the standard deviations (n=3).

Fig. 4.

V _max of Synechococcus sp. UTEX 2380 nitrate reductase (NR; A, B for kinetic phase I; C, D for kinetic phase II), nitrite reductase (NiR; E, F), and glutamine synthetase (GS; G, H). The data are expressed on a per cell basis in (A, C, E, G) and on a protein basis in (B, D, F, H). Different letters indicate significantly different means across all treatments (i.e. between different N sources and between N- and energy-limited cells).

Fig. 5.

Glutamine synthetase (GS) activity of NO₃⁻- (A, C) and NH₄⁺-grown (B, D) Synechococcus sp. UTEX 2380 cells as a function of sodium glutamate concentration. The activities were normalized by the number of cells (A, C) or by the amount of protein (B, D). The error bars indicate the standard deviations (n=3).

Fig. 6.

Expression of the nitrate reductase gene (narB) of Synechococcus sp. UTEX 2380 cultured under either N or energy limitation. Values are reported relative to the narB expression in energy-limited cells grown in NO₃⁻ at 550 μM. The error bars show the standard deviation (n=3). Different letters indicate significantly different means across all treatments (i.e. between different N sources and between N- and energy-limited cells) (P<0.05).

Fig. 7.

Nitrate assimilation in Synechococcus UTEX 2830 is regulated indirectly via 2-oxoglutarate (2OG), the carbon skeleton for glutamate synthesis, depending on intracellular NH₄⁺. In the presence of high concentration of NH₄⁺ (cell growth is limited by energy), 2OG is consumed, leading to the segregation of PipX–NtcA–2OG complex and the consequent inactivation of NtcA. This inactivation down-regulates or even completely blocks nitrate assimilation related (NAR) gene expression and nitrate utilization. On the contrary, when the growth is limited by N (irrespective of N source, energy is sufficient), intracellular 2OG accumulates and tends to bind to PII, inducing the release of PipX from the PII–PipX complex. PipX together with 2OG forms the PipX–NtcA–2OG complex, activating NtcA, and the active NtcA promotes NAR gene expression (nrtP, narB, and nirA also require NtcB) and nitrate assimilation. The internal NH₄⁺ is between these two extremes (high NH₄⁺ and N limitation) with sufficient NO₃⁻ present in the culture medium. 2OG, 2-oxoglutarate; Gln, glutamine; glnA, GSI gene; glnB, PII gene; Glu, glutamate; GSI, type I glutamine synthetase; GOGAT, glutamate synthase; narB, NR gene; NiR, nitrite reductase; nirA, NiR gene; NR, nitrate reductase; NrtP, nitrate/nitrite permease; nrtP, NrtP gene; NrtS1 and NrtS2 (NrtS1/S2), nitrate/nitrite bispecific transporter; nrtS1 and nrtS2, NrtS1 and nrtS2 gene; NtcA; global nitrogen regulator; ntcA, NtcA gene; NtcB, activator of NAR genes; ntcB, NtcB gene; PII, nitrogen regulation protein; PipX, interacting protein; pipX, PipX gene.