Sterivex gv

Cultures for exoproteome analysis were prepared by spinning the cells (3000 g at room temperature during 15 min) and gently re‐suspending them in fresh media at a final concentration of 3 × 10⁷ cell ml⁻¹. Cultures were then left to grow in standard conditions to cell densities of 10⁸ cells ml⁻¹. At this point, cultures were subjected to centrifugation at 3000 g for 15 min at room temperature. Supernatants were carefully removed and then gently filtered through 0.22 μm pore size filter units (Sterivex‐GV, Millipore) to eliminate any remaining cells. Proteins in the remaining milieu were concentrated and purified by precipitation with trichloroacetic acid and run on SDS‐PAGE as previously described (Christie‐Oleza and Armengaud, 2010). Trypsin in‐gel proteolysis of the entire exoproteome was performed for the shotgun proteomics analysis as recommended (Hartmann et al., 2014). NanoLC‐MS/MS experiments were performed using a LTQ‐Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex‐LC Packings). Conditions used were those previously described (de Groot et al., 2009).

Metaproteomic analysis of the Synechococcus sp. YX04-3 coculture was conducted to elucidate the molecular mechanisms underlying population-level interactions. The 22nd day of cocultivation was chosen for targeted proteomic analysis because the number of Synechococcus cells was equivalent to the number of heterotrophic bacterial cells at this time point. To generate the metaproteomes, triplicate 120-ml liquid cultures were centrifuged (3,000 × g for 15 min at 4°C). The supernatant was then filtered through 0.22-μm-pore-size filter unit (Sterivex-GV, Millipore) and subjected to exoproteome analysis. The detailed protocol was conducted by the method of Christie-Oleza et al. (8 (link)) and supplied in the supplemental material. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015067.

Samples from FGL, Fayetteville, NY, United States (43°3′N, 75°57.6″ W) were collected on August 14, 2013. We used a 12 L Niskin bottle to collect water samples in triplicates from five different depths (21, 24, 30, 45, and 52 m) and a grab corer to collect samples from the underlying sediments (∼53 m). Methane concentrations, pH values and temperatures measured at the time of sampling are provided in Supplementary Table 1. A permit to conduct sampling at FGL was obtained from the New York State Office of Parks, Recreation and Historic Preservation. Upon collection, all water samples were kept on ice for approximately 6 h and filtered through a 0.22 μm Sterivex GV filter (Millipore) using a Masterflex L/S 7553-70 peristaltic pump. Both filter samples and sediments were frozen at −80°C until DNA extraction. The anaerobic sediment slurry was kept at 4°C in the dark in tightly capped glass jars or serum bottles for later inoculations. Supplementary Table 2 presents all water column samples used in this study and their corresponding depths.