SE176:/S01/M02/D01

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	M02
Title	LC/MS/MS analysis
Method Details ID	MS02
Sample Amount
Comment

Analytical Method Details Information

ID	MS02
Title	LC/MS/MS analysis
Instrument	LCMS‐8040 triple quadrupole LC/MS/MS spectrometer; Shimadzu
Instrument Type
Ionization	ESI
Ion Mode
Description	Equal amounts of cells (10 ml cell culture with A730 = 1.0) were harvested by rapid filtration, and metabolites were extracted using a previously described method (Soga, 2007; Yoshida et al., 2008), with modifications. Briefly, the harvested cells were immediately filtered, and then the intermediate metabolites were quenched and extracted in a 1.2 ml solvent mixture (CHCl3 : CH3OH : H2O, 2.5:2.5:1, v/v/v) containing 10 μg l−1 of D-(+)-camphor-10-sulfonic acid as an internal standard for semiquantitative analysis. After centrifugation at 13 000 r.p.m. (15 000 g) at 4°C for 5 min, 400 μl of the upper phase was transferred to a new tube and vacuum-dried. The intracellular metabolites were quantified by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LCMS-8040 triple quadrupole LC/MS/MS spectrometer; Shimadzu, Kyoto, Japan) as described previously (Luo et al., 2007).
Comment_of_details

PubMed

PubMed

PubMed

Data Analysis Information

ID	D01
Title	Statistical analysis
Data Analysis Details ID	DS01
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS01
Title	Statistical analysis
Description	All statistical analyses were performed using StatPlus:macLE software for MacOSX (Analyst Soft, Springfield, VA, USA). Paired two-tailed Student's t-tests were performed to calculate P-values. A 95% confidence interval was used to determine significance.
Comment_of_details