UTurku - SufR-SRM

UTurku - SufR-SRM
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SufR-SRM_2015-10-21_12-50-22.sky.zip2017-02-17 11:05:189424424473218
Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses

  • Organism: Synechocystis sp. PCC 6803
  • Instrument: TSQ Vantage
  • SpikeIn: No
Background: Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Methods: The approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level. Results: The results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR. Conclusions: The study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism. General significance: The study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria.
Experiment Description
The SRM assays were performed on a TSQ Vantage QQQ mass spectrometer (Thermo Fischer Scientific) equipped with a nanoelectrospray ionization source. The desalted peptides were separated on a nanoflow HPLC system (EasyNanoLC 1000; Thermo Fisher Scientific). The injected amount of each unfractionated biological triplicate was 150 ng. A 60 min non-linear gradient (5-20% B in 35 min; 20–35% B in 50min; B = acetonitrile:water, 98:5) was applied at a 300 nL/ min flow rate. Once the peptides were eluted and ionized, they were analyzed in the QQQ-MS, operated in the SRM mode, as described (Vuorijoki et al., 2016). To maintain high sensitivity of the SRM measurements, the selected targets were divided to three different transition lists and scheduled assays with a 5 min retention time window for each peptide was applied, resulting in a cycle time of 2.5 s and a dwell time of >20 ms. The protein targets and the respective SRM assay parameters were selected from the public dataset, available in Panorama Public (https://panoramaweb.org/labkey/Vuorijoki_et_al_2015.url) (Vuorijoki et al., 2016). The data was processed with MSstats 3.1.4 and a global standard normalization was used with two endogenous peptides(SNLDSNHIIYR and SEELLGAASNR) of the probable DNA-directed RNA polymerase omega subunit (ssl2982).
Sample Description
Synechocystis sp. PCC 6803 sufR deletion mutant and the wild type strains were cultivated under photoautotrophic conditions in AlgaeTron AG 230 growth chamber (Photon Systems Instruments, Drásov, Czech Republic), under controlled, high CO2 conditions (1 % (vol/vol). The temperature was set to +30 °C and the light intensity to 50 µmol photons m-2 s-1. The cells were grown in BG-11 medium (Rippka et al., 1979), buffered with 20 mM TES-KOH (pH 8.0) in Erlenmeyer culture flasks on a rotary shaker (120 rpm). The precultures (40 ml BG-11 in 100 ml flasks) were grown under standard BG-11 media until mid-logarithmic phase, harvested by centrifugation and washed either one or three times in standard or iron depleted BG-11 media, respectively. The precultures were used to inoculate the main cultures (40 ml BG-11 in 100 ml flasks) to the starting OD750nm of 0.1. The cell density was estimated by measuring the optical density at 750 nm (OD750nm) with a Genesys 10S UV-VIS spectrophotometer (Thermo Scientific). The cells were harvested from both iron sufficient and deprived conditions at OD750nm 1.0, and after 12 days under iron deprivation as described in Vuorijoki et al 2016 (Vuorijoki et al., 2016).
Created on 2/17/17, 11:05 AM