Interaction of the Nitrogen Regulatory Protein GlnB (PII) with Biotin Carboxyl Carrier Protein (BCCP) Controls Acetyl-CoA Levels in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

The family of P_II signal transduction proteins (members GlnB, GlnK, NifI) plays key roles in various cellular processes related to nitrogen metabolism at different functional levels. Recent studies implied that P_II proteins may also be involved in the regulation of fatty acid metabolism, since GlnB proteins from Proteobacteria and from Arabidopsis thaliana were shown to interact with biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC). In case of Escherichia coli ACCase, this interaction reduces the k_cat of acetyl-CoA carboxylation, which should have a marked impact on the acetyl-CoA metabolism. In this study we show that the P_II protein of a unicellular cyanobacterium inhibits the biosynthetic activity of E. coli ACC and also interacts with cyanobacterial BCCP in an ATP and 2-oxoglutarate dependent manner. In a P_II mutant strain of Synechocystis strain PCC 6803, the lacking control leads to reduced acetyl-CoA levels, slightly increased levels of fatty acids and formation of lipid bodies as well as an altered fatty acid composition.

Keywords: BCCP; GlnB (PII); Synechocystis sp. PCC 6803; acetyl-CoA; cyanobacteria.

Figure 1

(A) Inhibition of ACC activity in response to various GlnB concentrations. The EC₅₀ value was calculated to be 0.31 μM (SE: 0.07 μM). (B) Inhibition of ACC with increasing 2-OG concentrations. The IC₅₀ was calculated to be 4.8 μM (SE: 0.2 μM). (C) Activity of ACC with various GlnB point mutated proteins present in the reaction mixture.

Figure 2

(A) Pull down experiments of Synechocystis BCCP and GlnB using BCCP as bait. Experiments were performed using 0.5 mM Mg²⁺, 0.5 mM ATP and 1 mM 2-OG where indicated. (B) ATP titration of the BCCP-GlnB interaction while the amount of GlnB used was held constant and a calculated EC₅₀ value of 68.0 μM (SE 13.2 μM) for ATP. The maximum binding was calculated to be 86.6 mpi (SE 3.8 mpi). (C) Influence of 2-OG on the BCCP-GlnB interaction. The IC₅₀ of 2-OG is 41.3 μM (SE 1.7 μM). (D) Increasing amounts of GlnB and its S49 variants were used with BCCP as bait with 0.5 mM Mg²⁺ and 0.5 mM ATP. The EC₅₀ is 65.9 nmol (SE 4.2 nmol) for wild type (circles), 77.0 nmol (SE 8.6 nmol) for S49C (triangles) and 224 nmol (SE 32.1 nmol) for S49D (inverted triangles). Maximum binding was calculated to be 100.7 mpi (SE 2.2 mpi) for the wild type protein, 91.4 mpi (SE 3.7 mpi) for the S49C variant and 21.0 mpi (SE 1.6 mpi) for the S49D variant. (E) ATP titration of the BCCP-GlnB S49C and S49D variant (as in B). The calculated ATP EC₅₀ value for S49C variant is 143 μM (SE 24 μM) and 231 μM (SE 36 μM) for the S49D variant. Maximum binding was calculated to be 88.2 mpi (SE 3.8 mpi) for the S49C variant and 32.2 mpi (SE 1.7 mpi) for the S49D variant.

Figure 3

Acetyl-CoA levels (A) and fatty acid levels (B) under different carbon nitrogen regimes during exponential growth in the wild type and the P_II mutant. Values represent the mean of three biological replicates. Differences in acetyl-CoA levels using the tested growth conditions are statistically significant (p < 0.05; unpaired t-test). Statistically significant values of fatty acid levels are marked with a star (p < 0.05; unpaired t-test).

Figure 4

Accumulation of acetyl-CoA (A) and fatty acids (B) during growth in standard BG11 with ammonia as nitrogen source in wild type (white bars), the P_II mutant (gray bars), the P_II complemented strain (checked bars) and the P_II-S49E complemented strain (gray dotted bars). Values represent the mean of three biological replicates. Differences in acetyl-CoA levels are statistically significant throughout growth between wild type and the P_II mutant (p < 0.05; unpaired t-test). Differences in fatty acid levels between wild type and the P_II mutant are statistically significant within the first 48 h of growth (p < 0.05; unpaired t-test).

Figure 5

(A) Microscopic image of wild type and P_II mutant stained with Bodipy 493/503 during exponential growth. Images are the superimposition of the fluorescence and bright field image. (B) Occurrence of lipid droplets per cell in the wild type (mean: 0.39 ± 0.12) and P_II mutant (mean: 1.65 ± 0.23) at an OD₇₅₀ of 0.2. The box plot displays the 10–90 percentile. The result is statistically significant with a p-value smaller than 0.0001 determined by an unpaired two tailed t-test. (C) Thin layer chromatography of hydrophobic lipids. Possible triacylglycerols are marked with asterisk. DAG: diacylglycerol TAG: triacylglycerol.