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Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803
NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane, unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that NdbA, which accumulates in WT thylakoid membrane particularly under LAHG conditions, has a function in redox sensing and regulation, and ultimately fine-tunes the gene expression in Synechocystis cells to meet the requirements for optimal growth under LAHG conditions.
Sample Processing Protocol
Cells were collected by centrifugation at 8000 × g for 10 min at 4 °C and washed twice with 50 mM TES-KOH buffer, pH 8.0. The cell pellet was suspended in a protein extraction buffer (0.1 % (w/v) RapiGest SF (Waters Corporation, Milford, MA) in 8 M urea solubilized in 0.1 M Tris-HCl supplemented with 200 µM PMSF) together with an equal volume of acid washed glass beads (150-212 µm, Sigma). The cells were thereafter broken using a bead beater (Mini-Bead-Beater-8, Unigenetics Instruments Pvt. Ltd., India) with six cycles of 45 s shake with 3 minutes incubation on ice. Unbroken cells and glass beads were removed by centrifugation for 5 min at 1000 x g and again for 20 min at 12000 x g at room temperature. The protein concentration was measured with the Bradford method (Bio-Rad). Proteins were reduced with 5 mM dithiotreitol (DTT, Sigma), alkylated with 10 mM iodoacetamide (IAA, Sigma) and precipitated with 1:5 v/v of 50% acetone/ 50 % ethanol o/n at -20 °C. The pellet was centrifuged at 13,200 rpm at +4 °C for 30 min and solubilized by trypsin (Sequence grade Modified, Promega, Madison, WI, USA) digestion (1:100 trypsin:protein ratio) in 50 mM Tris-HCl and 5 % (v/v) acetonitrile (ACN) buffer for 4-5 hours at +37 °C shaking. Digestion was continued for an additional 15-16 hours following a second addition of trypsin in the same ratio. The digestion was stopped by adding formic acid (FA) (Sigma) to a final concentration of 0.5–1 % (v/v) to lower the pH below 2. The digestion mixture was incubated for 30 min at +37 °C at 130 rpm and the water immiscible RapiGest SF degradation products were removed by centrifugation. To desalt the samples, solid phase extraction (SPE) of the peptide mixture was performed with Wet Sep-Pak 100 mg C18 96 well plate according to manufacturer’s protocol. The eluted peptide samples were vacuum-dried (Savant SPD1010, SpeedVac Concentrator, Thermo Scientific) and solubilized in 0,1 % (v/v) FA and 2 % (v/v) ACN and stored at -80 °C prior to MS analysis. MS data was acquired automatically by using Thermo Xcalibur 3.1 software (Thermo Fisher Scientific). A data dependent acquisition method consisted of an Orbitrap MS survey scan of mass range 300-1800 m/z followed by HCD fragmentation for 12 most intense peptide ions. The spectra were registered with a resolution of 120000 and 15000 (at m/z 200) for full scan and for fragment ions, respectively, and normalized collision energy of 27 %. The AGC (automatic gain control) was set to a maximum fill time of 100 ms and 250 ms to obtain maximum number of 3e6 and 1e5 ions for MS and MS/MS scans, respectively
Data Processing Protocol
Data files were searched for protein identification against Synechocystis database retrieved from Cyanobase (Kaneko et al., 1996) (3672 entries, 23.10.2012) using Proteome Discoverer 2.2 software (Thermo Fisher Scientific) connected to an in-house server running the Mascot 2.6.1 (Perkins et al., 1999) algorithm (Matrix Science). The precursor value was restricted to monoisotopic with a mass tolerance of ±4 ppm and fragment ion mass tolerance of ±0.02 Da. Two missed cleavages were allowed and decoy searches were performed. For the validation of the spectrum identifications, we used a Percolator algorithm (Käll et al., 2007) with relaxed false discovery rate (FDR) of 0.05. The original data and protein identification files are deposited in PRIDE Archive database (Vizcaíno et al., 2016). Quantitative analysis was done in Progenesis software with global normalization and using relative quantification of proteins with at least two peptides with no conflicts per protein.
Huokko T, Muth-Pawlak D, Aro EM. Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803. Plant Cell Physiol. 2019 PubMed: 30847494
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