NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis) the exact roles of different NDH2s remain poorly understood. To elucidate this question we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (ΔndbC) for corresponding protein and submitting the mutant to physiological and biochemical characterisation as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the ΔndbC mutant under light activated heterotrophic growth conditions but not under mixotrophy. Thus NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth condition.

The target ndbC lacking Synechocystis sp. PCC 6803 strain was cultivated under the ambient CO2 conditions. The proteins were extracted and digested and the peptides mixtures were subjected to shotgun LC-MS/MS analysis (QExactive mass spectrometer). The obtained data was searched against S. 6803 proteome and the combined results were used to generate spectral library in Skyline. SRM-assays used in the experiment were downloaded from Vuorijoki et al 2016 or developped according the same protocol for the new targets. We were able to reliably detect (TSQ Vantage) and quantify 105 proteins in unfractionated cell lysates.

The strains used in this study were Synechocystis sp. PCC 6803 (WT) and the ΔndbC mutant (constructed in this study). Pre-experimental cultures were maintained in BG11 medium buffered with 20mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, (HEPES-NaOH, pH 7.5) under continuous illumination of 50 µmol photons m-2 s-1 (PAR), 3% CO2, 30 °C with agitation of 150 rpm. All experimental cultures were grown in AlgaeTron AG130 growth chambers (PSI Instruments, Czech) under continuous illumination of 50 μmol photons m-2 s-1 (cool-white LED). . During experimental cultivation cells were grown in +30 °C with agitation of 150 rpm. Cells were inoculated to OD750nm=0.1 and grown for 72 h in ambient CO2 (LC) conditions before collecting samples. Total proteins were isolated from cell cultures as described in Zhang et al. (2009). Equivalent of 30 µg total protein was mixed with 2 x Laemmli buffer (with 8M urea) in 1:1 ratio and loaded on a 12% (50% acrylamide, 1.3% bis-acrylamide) stacking gel (0.5 M Tris-HCl, pH 6.8) containing 6 M urea, no SDS. Gel bands containing all proteins were reduced, alkylated and digested according to protocol described by Shevchenko et al. (1996,2006) (Shevchenko et al., 1996) (Shevchenko et al., 2006). Extracted peptides were vacuum dried and further solubilized in 2% acetonitrile (ACN), 0.1% formic acid (FA) just before MS analysis. Samples were prepared in 4 biological replicates from WT and the ΔndbC mutant.