Rocha E R, Selby T, Coleman J P, Smith C J
Department of Microbiology and Immunology, School of Medicine, East Carolina University, Greenville, North Carolina, USA.
J Bacteriol. 1996 Dec;178(23):6895-903. doi: 10.1128/jb.178.23.6895-6903.1996.
Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.
脆弱拟杆菌在有氧环境中的存活取决于细菌合成新蛋白质的能力，这是通过暴露于氧气后蛋白质合成受到抑制来确定的。在有无百草枯的情况下从厌氧条件转变为有氧条件或添加外源过氧化氢后，脆弱拟杆菌的蛋白质谱发生了显著变化。通过二维凝胶电泳后的放射自显影测定，在氧化条件下检测到约28种新合成的蛋白质。这些蛋白质的等电点值（从5.1到7.2）和分子量（从12,000到79,000）范围很广。从二维凝胶蛋白质谱来看，过氧化氢和百草枯诱导的反应与氧气诱导的反应相似但不完全相同。其中11种氧化反应蛋白密切相关，其等电点值分别为5.1至5.8，分子量为17,000至23,000。作为理解对氧气抗性的第一步，通过等位基因交换构建了一个过氧化氢酶缺陷型突变体。当向培养基中添加亚铁螯合剂联吡啶时，发现katB突变体比亲本菌株对过氧化氢的致死作用更敏感。这表明厌氧培养基中亚铁的存在会加剧过氧化氢的毒性，并且功能性过氧化氢酶的存在对于在过氧化氢存在下的存活很重要。此外用亚致死浓度的过氧化氢处理培养物对于诱导亲本菌株对更高浓度的过氧化氢产生抗性是必要的，这表明这是一种可诱导的反应。当在细胞暴露于亚致死浓度的过氧化物之前用氯霉素处理细菌培养物时，细胞完全丧失活力，这证实了这一点。相比之下，当过氧化物诱导后发生蛋白质合成抑制时细胞活力得到了极大的保留。突变体中过氧化氢酶活性的互补恢复了突变菌株在过氧化氢存在下的存活能力，表明过氧化氢酶（KatB）可能在耐氧厌氧菌的氧化应激抗性中发挥作用。
