The M. acetivorans gene expression data (Figures 1, 2, 3, 4, 5, 6, 7, 8) provides a foundation to understand how energy-yielding pathways are regulated in this model organism and in related methanogens. It is unknown if this control occurs by the actions of classical transcription factors like those found in bacteria and eukaryotes, and/or by RNA control mechanisms involving attenuation, regulated termination and/or small RNAs. Methods Cell culture Methanosarcina acetivorans

C2A [1] was cultivated in a mineral medium that contained (in grams per liter): NaCl, 11.69 g; MgSO4 7H2O, 12.32 g; KCl, 0.76 g; CaCl2·2H2O, 0.14 g; NH4Cl, 0.5 g; Resazurin solution (10,000 × stock solution), 0.1 ml; trace metal solution (100×) 10 ml [29]; vitamin solution (100×) 10 ml [29]; HCl (12.1 N) 0.5 ml; Na2HPO4 7H2O, 1.12 PF-01367338 nmr g; cysteine-HCl H2O, 0.25 g; Na2CO3, 3.0 g. An atmosphere (80:20) of nitrogen to carbon dioxide was used in the vessel headspace. Following sterilization, the medium was supplemented with filter-sterilized 0.1 ml 50% methanol or 0.2 ml 5 M acetate per 10 ml medium as previously Alvocidib described [30].

RNA purification For RNA isolation, cultures of M. acetivorans C2A cells were grown on acetate or methanol with serial transfer of three times to mid-exponential phase before cell harvest. Total RNA was purified from 10 ml of cell samples using the RNAwiz (Ambion Austin, TX) following the manufacturer’s instructions.

The purified RNA was treated with DNase I as described [31, 32]. Quantitative RT-PCR The real time reverse selleck inhibitor transcription (RT-PCR) reactions were performed using Superscript II reverse transcriptase (Invitrogen Carlsbad, CA) according Erlotinib research buy to the manufacturers recommended protocol using random primers and 1 μg of total RNA. A mock reaction without Superscript was run to evaluate for the presence of genomic DNA contamination. To remove complementary RNA, 1 μl RNase H was added to mixture and incubated for 20 min at 37°C. The RNase was then heat inactivated at 70°C for 15 min. The cDNA from the RT reaction was diluted 10 fold, and 1 μl of the diluted cDNA was subsequently used in a 30 μl iQ SYBR green supermix according to the manufactures recommendations following addition of 1.5 μl DMSO. The real time PCR reactions were conducted on a Biorad iCycler (Biorad, Hercules, CA) or an Eppendorf Realtime2 (Eppendorf, Westbury, NY) using a four-step program consisting of, denaturing, annealing, extension, and acquisition steps. The RT-PCR primers were created by a modified version of MyPROBES [32]. The PCR product lengths were in a range of 100-200 bp, the melting temperature was in the range of 55-66°C, the GC content was 55-65%, and the primer length was 17-22 bases (Additional file 4, Table S1). The primers were tested against serial dilution of genomic DNA (106 to 102 copies) to generate a standard curve for each gene tested.