Steady-state samples of Methanococcus maripaludis MM901:del_mtd (MMP0372). Methanococcus maripaludis S2

Effects of mtd (MMP0372) on physiology and regulation of methanogenesis in Methanococcus maripaludis Overall design: The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37°C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). Hydrogen limited condition was 20 mL/min H2. After the OD increased above 0.6 (24 h), the medium flow was turned on at 0.083 L/h. Medium was either phosphate limiting (0.12 mM K2HPO4) or phosphate excess (0.8 mM K2HPO4). Culture samples (1.5 mL) were rapidly removed from the vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80°C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

马氏甲烷球菌（Methanococcus maripaludis）中mtd基因（MMP0372）对其生理特性及产甲烷过程调控的影响

实验总体设计：该菌株采用1升发酵罐（New Brunswick Scientific，美国新泽西州爱迪生市）于37°C下进行连续培养，培养方法参考已发表文献（FEMS Microbiol Lett 238: 85-91, 2004）。培养基与气体组分均基于非限培养条件的标准配方进行优化调整，基础配方参考已发表文献（BMC Microbiol 9: 149, 2009）。标准通气方案为：氢气110 mL/min、二氧化碳40 mL/min、氩气35 mL/min，以及15 mL/min的H2S/Ar混合气（体积比1:99）；氢限制培养条件下的通气方案调整为氢气20 mL/min。当光密度（OD）值升至0.6以上（约培养24小时）后，以0.083 L/h的流速开启培养基补料。培养基分为两种类型：磷限制型（磷酸氢二钾浓度为0.12 mM）与磷过量型（磷酸氢二钾浓度为0.8 mM）。采用注射器从发酵罐中快速采集1.5 mL培养物样本，通过微量离心收集细胞沉淀，随后立即置于乙醇-干冰浴中冷冻，并保存于-80°C冰箱。

提取每份样本的总RNA，并以批量培养的MM901菌株对数中期培养物制备的参考RNA混合池作为对照样本，用于与各样本的总RNA进行比较。将各样本与参考样本的总RNA分别直接用Cy3或Cy5荧光染料标记，随后与平铺式微阵列芯片（tiling array）进行杂交。按照芯片制造商的说明书完成杂交与洗涤步骤后，使用微阵列扫描仪（Microarray Scanner，安捷伦科技公司，美国加利福尼亚州圣克拉拉市）对芯片进行扫描。针对每份样本均开展染料互换实验（dye-flip）。