SE177:/S01/M01/D01

Sample Set Information

ID	TSE1335
Title	Metabolomic analysis reveals rewiring of Synechocystis sp. PCC 6803 primary metabolism by ntcA overexpression.
Description	NtcA is a cAMP receptor protein-type transcription factor conserved among cyanobacteria and is essential for gene expression in response to nitrogen status. NtcA has been widely studied; however, no metabolomic analysis has been conducted using the ntcA mutant. Here, we generated a strain that overexpresses ntcA in Synechocystis sp. PCC 6803, named NOX10, and performed physiological, transcriptomic and metabolomic analyses. NOX10 grew faster than the wild-type strain under photoautotrophic conditions, but slower under light-activated heterotrophic conditions. Transcriptome analysis revealed that the expression of genes related to primary metabolism was altered by ntcA overexpression particularly under nitrogen-depleted conditions. Metabolomic analysis revealed that metabolite levels in sugar, purine/pyrimidine nucleotide, organic acid and amino acid metabolism were widely altered by ntcA overexpression. The protein levels of nitrogen-regulated transcriptional regulators were altered by ntcA overexpression during nitrogen starvation. These results demonstrate the alteration of primary metabolism by genetic engineering of NtcA, and they contribute to the current understanding of metabolic regulation of unicellular cyanobacteria.
Authors	Osanai, T., Oikawa, A., Iijima, H., Kuwahara, A., Asayama, M., Tanaka, K., Ikeuchi, M., Saito, K. and Hirai, M.Y.
Reference	Environ Microbiol. 2014 Oct;16(10):3304-17. doi: 10.1111/1462-2920.12554.
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	Cultivation of cyanobacterial strains was performed as previously described (Osanai et al., 2013a). The GT strain of Synechocystis sp. PCC 6803, isolated by Williams (1988), was grown in modified BG-11 medium (Rippka, 1988), containing 5 mM NH4Cl, instead of 17.5 mM NaNO3 (buffered with 20 mM Hepes-KOH, pH 7.8). Of 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). 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

PubMed

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	Cell collection and metabolite extraction for CE‐MS were performed as previously described (Osanai et al., 2014b). The CE‐MS system and conditions were as described previously (Oikawa et al., 2011).
Comment_of_details

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, Fairfax, 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