Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ factors of Arcobacter butzleri

The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintainance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation. Overall design: Eleven condition experiment, wild-type A. butzleri and 10 mutants. Three replicates for isolates RM4810, RM4810∆Aσ1::Km, RM4018Δσ1/Aσ1::Km, RM4810∆Aσ2::Km, RM4018∆σ2/Aσ2::Km, RM4018∆Aσ4::Km, RM4018∆σ4/Aσ4::Km, RM4018∆Aσ5::Km, RM4018∆σ5/Aσ5::Km, RM4018∆Aσ7::Km, and RM4018Δσ7/Aσ7::Km independently grown in BHI and incubated at 30°C on a shaking platform set at 150 rpm. RNA was isolated from 5 ml of mid-logarithmic phase cultures (OD550 of approximately 0.5). A type 2 gene expression experimental design was used, with fluorescently labeled genomic DNA as a reference channel in each experiment as described by Lucchini, S., et al. 2005. Infect Immun 73:88-102.胞质外功能（extracytoplasmic function, ECF）σ因子是细菌适应不同环境、在多种胁迫条件下存活的关键调控元件。新兴病原菌布氏弓形杆菌（Arcobacter butzleri）基因组中存在7套隶属于ECF家族的推定σ/抗σ因子对。本研究鉴定了该菌7种ECF σ因子中5种所调控的靶基因。其中3种ECF σ因子在物质转运、能量生成及氧化还原平衡维持中发挥显著功能。诸如nap、sox与tct等基因可被多种ECF σ因子共同调控，提示布氏弓形杆菌的ECF σ因子构成了重叠调节子网络。与其他真细菌不同，布氏弓形杆菌的ECF调节子似乎主要通过调控环境响应以满足代谢需求，而非在胁迫适应中承担核心功能。 实验整体设计：本实验共设置11个实验组：野生型布氏弓形杆菌与10株突变菌株。对RM4810、RM4810∆Aσ1::Km、RM4018Δσ1/Aσ1::Km、RM4810∆Aσ2::Km、RM4018∆σ2/Aσ2::Km、RM4018∆Aσ4::Km、RM4018∆σ4/Aσ4::Km、RM4018∆Aσ5::Km、RM4018∆σ5/Aσ5::Km、RM4018∆Aσ7::Km及RM4018Δσ7/Aσ7::Km均设置3次生物学重复，所有菌株均接种于脑心浸液培养基（Brain Heart Infusion, BHI）中，于30℃、摇床转速150 rpm条件下培养。从5 mL对数生长期中期（OD550约为0.5）的培养物中提取总RNA。本研究采用第二类基因表达实验设计，以荧光标记基因组DNA作为每个实验的参考通道，具体实验方法参照Lucchini S.等人2005年发表于Infect Immun 73:88-102的研究方案。