SE180:/S01/M03

Sample Set Information

ID	TSE1338
Title	Alteration of cyanobacterial sugar and amino acid metabolism by overexpression hik8, encoding a KaiC-associated histidine kinase.
Description	Cyanobacteria possess circadian clocks consisting of KaiABC proteins, and circadian rhythm must closely relate to the primary metabolism. A histidine kinase, SasA, interacts with KaiC to transduce circadian signals and widely regulates transcription in Synechococcus sp. PCC 7942, although the involvement of SasA in primary metabolism has not been demonstrated at metabolite levels. Here, we generated a strain overexpressing hik8 (HOX80), an orthologue of SasA in Synechocystis sp. PCC 6803. HOX80 grew slowly under light conditions and lost viability under continuous dark conditions. Transcript levels of genes related to sugar catabolism remained higher in HOX80 under dark conditions. Metabolomic analysis revealed that under light conditions, glycogen was undetectable in HOX80, and there were decreased levels of metabolites of sugar catabolism and increased levels of amino acids, compared with those in the wild-type strain. HOX80 exhibited aberrant degradation of SigE proteins after a light-to-dark transition and immunoprecipitation analysis revealed that Hik8 directly interacts with KaiC1. The results of this study demonstrate that overexpression of hik8 widely alters sugar and amino acid metabolism, revealing the involvement of Hik8 in primary metabolism under both light and dark conditions in this cyanobacterium.
Authors	Osanai, T., Shirai, T., Iijima, H., Suzuki, I., Kondo, A. and Hirai, M.Y.
Reference	Environ Microbiol. 2015 Jul;17(7):2430-40. doi: 10.1111/1462-2920.12715
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 strain of Synechocystis sp. PCC 6803, isolated by Williams (1988), and the hik8‐overexpressing strain, designated as HOX80, were grown in modified BG‐11 medium, which consisted of BG‐110 liquid medium (Rippka, 1988) containing 5 mM NH4Cl (buffered with 20 mM Hepes‐KOH, pH 7.8). Among 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 and incubated at 30°C under continuous white light (c. 50–70 μmol photons m−2 s−1). For plate cultures, modified BG‐11 medium (containing 10 mM NH4Cl in liquid medium) was solidified with agar (1.5% w/v), and cultures were incubated in air at 30°C under continuous white light (c. 50–70 μmol photons m−2 s−1). Growth and cell densities were measured at OD730 with a Hitachi U‐3310 spectrophotometer (Hitachi High‐Tech., Tokyo, Japan).
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M03
Title	Organic acid analysis by GC‐MS
Method Details ID	MS03
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS03
Title	Organic acid analysis by GC‐MS
Instrument	GCMS‐QP2010 Ultra
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	Equal amounts of cells (10 ml cell culture with OD730 = 1.0) were harvested by rapid filtration using a previously described method (Osanai et al., 2014a). GC‐MS was carried out using a GCMS‐QP2010 Ultra equipped with a CP‐Sil 8 CB‐MS capillary column (30 m × 0.25 mm × 0.25 μm; Agilent, Palo Alto, CA, USA). Helium was used as the carrier gas. The flow rate was 2.1 ml min−1. The injection volume was 1 μl with a split ratio of 1:10. An initial oven temperature of 60°C was maintained for 10 min, then raised to 315°C at 15°C min−1, and maintained for 6 min. The total running time was 33 min. The other settings were as follows: 250°C interface temperature, 200°C ion source temperature and electron impact ionization at 70 eV. Organic acids were derivatized prior to analysis. The dried residue was derivatized for 90 min at 30°C in 20 μl 20 mg ml−1 methoxyamine hydrochloride in pyridine. Subsequently, trimethylsilylation (TMS derivatization) was performed for 30 min at 37°C and then for 2 h at room temperature with 50 μl N‐methyl‐N‐(trimethylsilyl)trifluoroacetamide (Roessner et al., 2000; Strelkov et al., 2004).
Comment_of_details

PubMed