Elsevier

Journal of Plant Physiology

Volume 205, 20 October 2016, Pages 105-112
Journal of Plant Physiology

Metabolomic analysis of NAD kinase-deficient mutants of the cyanobacterium Synechocystis sp. PCC 6803

https://doi.org/10.1016/j.jplph.2016.09.002Get rights and content

Abstract

NAD kinase (NADK) phosphorylates NAD(H) to NADP(H). The enzyme has a crucial role in the regulation of the NADP(H)/NAD(H) ratio in various organisms. The unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses two NADK-encoding genes, sll1415 and slr0400. To elucidate the metabolic change in NADK-deficient mutants growing under photoautotrophic conditions, we conducted metabolomic analysis using capillary electrophoresis mass spectrometry (CE-MS). The growth curves of the wild-type parent (WT) and NADK-deficient mutants (Δ1415 and Δ0400) did not show any differences under photoautotrophic conditions. The NAD(P)(H) balance showed abnormality in both mutants. However, only the metabolite pattern of Δ0400 showed differences compared to WT. These results indicated that the two NADK isoforms have distinct functions in cyanobacterial metabolism.

Introduction

Pyridine nucleotides are cofactors involved in numerous redox reactions in all organisms. NAD+ and NADH mainly function in catabolic reactions, while NADP+ and NADPH participate in anabolic reactions and defenses against oxidative stress (Outten and Culotta, 2003, Pollak et al., 2007, Ziegler, 2000). Notably, NADP+ is converted to NADPH by the donation of electrons from photosystem I through ferredoxin, and by the oxidative pentose phosphate pathway.

NAD kinase is the NADP+ biosynthetic enzyme that regulates the balance between NAD(H) and NADP(H) (Ohashi et al., 2011). Genes encoding NAD kinase have been identified from many organisms, such as Homo sapiens (Lerner, 2001), Escherichia coli (Kawai et al., 2001a), Saccharomyces cerevisiae (Kawai et al., 2001b, Outten and Culotta, 2003), and Arabidopsis thaliana (Turner et al., 2004). Functional analyses have been performed in these organisms (Hashida et al., 2009, Kawai et al., 2000, Raffaelli et al., 2004, Sakuraba et al., 2005). In A. thaliana, three NAD kinase-encoding genes (NADK1, NADK2, and NADK3) have been identified (Berrin et al., 2005, Turner et al., 2005). NADK1 localizes to the cytosol (Chai et al., 2006). NADK2 is a chloroplast-localizing enzyme that can bind to calcium/calmodulin; this version is known to play a vital role in energy transduction through photosynthetic electron transport (Chai et al., 2005, Takahashi et al., 2006, Turner et al., 2004). NADK3 has been reported to localize to the peroxisomal matrix, where the enzyme serves as a source of NADPH (Waller et al., 2010). In our previous study, we demonstrated that alteration of the NAD/NADP balance in higher plants affected metabolism and reactive oxygen species (ROS)—specific responses (Takahara et al., 2010, Takahashi et al., 2009). Specifically, NADK2-overexpressing rice plants exhibited increased photosynthetic electron transport and CO2 assimilation rates, as well as enhanced tolerance to oxidative stress (Takahara et al., 2010).

Cyanobacteria have flexible metabolic capabilities, permitting these microbes to adapt to various environments. Cyanobacteria have garnered interest as potential renewable sources of biomass for sustainable production of biofuels (Dismukes et al., 2008, Mussatto et al., 2010, Wijffels et al., 2013). Several species of cyanobacteria possess the ability to store large amount of glycogen and lipids that can be utilized for the synthesis of bioethanol (Aikawa et al., 2013, Aikawa et al., 2014). Furthermore, cyanobacteria have been investigated in wide ranging metabolic flux and −omics analyses (You et al., 2015), including their potential use for the over-production of hydrogenases that might find application in industrial hydrogen synthesis (Belkin and Padan, 1978).

Synechocystis sp. PCC 6803 is a useful model species. The PCC 6803 genome includes two genes (sll1415 and slr0400) that encode putative NAD kinases (Gao and Xu, 2012). It was found that both sll1415 and slr0400 included conserved amino acid sequence motifs for NAD binding (GGDG) (Mori et al., 2005, Raffaelli et al., 2004) and ATP binding (NE/D) (Liu et al., 2005). Based on BLAST searches of the cyanobacterial genomes available in the NCBL GenBank database, all cyanobacteria harbor two types of NADK genes, and each NAD kinase-encoding paralog belongs to either the sll1415 or slr0400 clade (Gao and Xu, 2012). Gao and Xu (2012) demonstrated that PCC 6803 strains mutated for either NAD kinase-encoding gene show distinct sensitivities to methyl viologen. As we show in the present work, metabolomic analysis of NADK-deficient mutants of PCC 6803 revealed that the metabolite patterns differ between two NADK mutants under photoautotrophic conditions.

Section snippets

Strain and culture conditions

In this study, we used Synechocystis sp. PCC 6803 as the wild-type strain (WT). All strains were grown in BG11 culture medium (20 mL) on a shaker (100 rpm) at 30 °C under continuous light at 30 μE/m2/s. To obtain NADK-deficient mutants (Δ1415 and Δ0400), antibiotic resistant cassettes (encoding spectinomycin resistance for Δ1415 or kanamycin resistance for Δ0400) were inserted by homologous recombination into the respective NADK gene. After selection of antibiotic-resistant colonies, segregation

Analysis of NADK-deficient mutants of Synechocystis sp. PCC 6803

In this study, we constructed two NADK mutants (Δ1415 and Δ0400) in order to clarify the biological function of the two NADKs in Synechocystis sp. PCC 6803. As shown in Fig. 1A, the Δ1415 strain harbored an insertion of a spectinomycin resistance-encoding cassette into the sll1415 gene; the Δ0400 strain harbored an insertion of a kanamycin resistance-encoding cassette into the slr0400 gene. Using primers shown in Fig. 1A, the stability of each strain was confirmed by segregation analysis (Fig. 1

Discussion

In this study, we performed metabolomic analysis to clarify effects of NADK on metabolism in Synechocystis sp. PCC 6803. First, we assessed NADK activity and growth rate in NADK mutants (Δ1415 and Δ0400) growing under photoautotrophic conditions. Notably, Δ1415 showed a 48.7% decrease in NADK activity compared to WT. On the other hand, Δ0400 was not significantly altered in NADK activity. Furthermore, neither single mutant exhibited growth abnormality under photoautotrophic conditions,

Acknowledgement

The present study was supported by JSPS KAKENHI Grant Numbers 26292190 & 15K07094.

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