SE176:/S01/M01

Sample Set Information

ID	TSE1334
Title	Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803.
Description	Nitrogen availability is one of the most important factors for the survival of cyanobacteria. Previous studies on Synechocystis revealed a contradictory situation with regard to metabolism during nitrogen starvation; that is, glycogen accumulated even though the expressions of sugar catabolic genes were widely upregulated. Here, we conducted transcript and metabolomic analyses using capillary electrophoresis-mass spectrometry on Synechocystis sp. PCC 6803 under nitrogen starvation. The levels of some tricarboxylic acid cycle intermediates (succinate, malate and fumarate) were greatly increased by nitrogen deprivation. Purine and pyrimidine nucleotides were markedly downregulated under nitrogen depletion. The levels of 19 amino acids changed under nitrogen deprivation, especially those of amino acids synthesized from pyruvate and phosphoenolpyruvate, which showed marked increases. Liquid chromatography-mass spectrometry analysis demonstrated that the amount of NADPH and the NADPH/NADH ratio decreased under nitrogen depletion. These data demonstrate that there are increases in not only glycogen but also in metabolites downstream of sugar catabolism in Synechocystis sp. PCC 6803 under nitrogen starvation, resolving the contradiction between glycogen accumulation and induction of sugar catabolic gene expression in this unicellular cyanobacterium.
Authors	Osanai, T., Oikawa, A., Shirai, T., Kuwahara, A., Iijima, H., Tanaka, K., Ikeuchi, M., Kondo, A., Saito, K. and Hirai, M.Y.
Reference	Environ Microbiol. 2014 Feb;16(2):512-24. doi: 10.1111/1462-2920.12170.
Comment

PubMed

Sample Information

ID	S01
Title	Synechocystis sp. PCC 6803
Organism - Scientific Name	Synechocystis sp. PCC 6803 substr. GT-I
Organism - ID	NCBI:txid1080228
Compound - ID
Compound - Source
Preparation	The glucose‐tolerant (GT) strain of Synechocystis sp. PCC 6803, isolated by Williams (1988), was grown in modified BG‐11 medium (Rippka, 1988), which contains 5 mM NH4Cl, instead of 17.5 mM NaNO3 (buffered with 20 mM Hepes‐KOH, pH 7.8). Among the GT substrains, the GT‐I strain was used in this study (Kanesaki et al., 2012). Liquid cultures were bubbled with 1% (v/v) CO2 in air at 30°C under continuous white light (approximately 50–70 μmol photons m−2 s−1). For plate cultures, cyanobacteria were grown on modified BG‐110 (containing 10 mM NH4Cl, instead of 5 mM NH4Cl in liquid medium) solidified with 1.5% (w/v) agar, and incubated in air at 30°C under continuous white light (approximately 50–70 μmol photons m−2 s−1). Growth and cell densities were measured at A730 with a Hitachi U‐3310 spectrophotometer (Hitachi, Tokyo, Japan). To create nitrogen‐starved conditions, cells were collected by filtration with mixed cellulose ester (Advantec, Tokyo, Japan) and resuspended in BG‐110 liquid medium.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M01
Title	CE/MS analysis
Method Details ID	MS01
Sample Amount	20 μl
Comment

Analytical Method Details Information

ID	MS01
Title	CE/MS analysis
Instrument	CE:Agilent CE capillary electrophoresis system (Agilent Technologies) TOF-MS:Agilent G3250AA LC/MSD TOF system (Agilent Technologies) CE-MS:Agilent G1603A
Instrument Type
Ionization	ESI
Ion Mode	positive and negative
Description	Capillary electrophoresis–mass spectrometry was performed as described by Osanai and colleagues (2011). Cells of mid‐exponential phase cultures of Synechocystis 6803 (A730, 0.5–0.7) grown in modified BG‐11 or BG‐110 medium were collected by centrifugation at 10 000 r.p.m. (9000 g) for 2 min, and then frozen in liquid nitrogen. Cells (50–100 mg fresh weight) were suspended in 600 μl 60% (v/v) methanol containing 200 μM each of 10‐camphorsulfonic acid and trimesic acid as internal standards, and mixed using an MT‐200 microtube mixer (Tomy, Tokyo, Japan) eight times for 1 min at 4°C, and then centrifuged at 20 400 g for 5 min at 4°C. An aliquot (300 μl) of the supernatant was centrifuged through a Millipore 5‐kD cut‐off filter (Merck, Billerica, MA, USA) at 10 000 r.p.m. (9000 g) for 90 min, and 250 μl of the filtrate was dried for 120 min in a centrifugal concentrator. The residue was dissolved in 20 μl of water and subjected to CE/MS analysis. The CE/MS system and conditions were as previously described (Oikawa et al., 2011).
Comment_of_details

PubMed