SE177:/S01/M02

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	M02
Title	GC‐MS analysis
Method Details ID	MS02
Sample Amount	1 μl
Comment

Analytical Method Details Information

ID	MS02
Title	GC‐MS analysis
Instrument	GCMS‐QP2010Plus (Shimadzu)
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	Equal amounts of cells (50 ml of cell culture with A730 = 1.0) were harvested by rapid filtration using a previously described method (Osanai et al., 2014b). In total, 300 μl of the upper phase was transferred to a new tube and vacuum dried. Samples were suspended in 500 μl methanol with 1 μM norvaline as an internal standard and derivatized using the EZ:faast kit (Phenomenex, CA, USA) (Badawy et al., 2008). GC‐MS was performed using GCMS‐QP2010Plus (Shimadzu, Kyoto, Japan) equipped with a 10 m × 0.25 mm ZB‐AAA capillary GC column. The injection volume was 1 μl at a carrier gas flow of 1.17 ml min−1 helium with a split ratio of 1:15. The initial column oven temperature of 110°C was maintained for 1 min and raised to 300°C at 20°C min−1. The interface and ion source temperatures were 280°C and 240°C respectively.
Comment_of_details