Moshiri F, Kim J W, Fu C, Maier R J
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Mol Microbiol. 1994 Oct;14(1):101-14. doi: 10.1111/j.1365-2958.1994.tb01270.x.
The FeSII protein of Azotobacter vinelandii has been proposed to mediate the 'conformational protection' of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII locus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesII locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is upregulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other sigma 70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCl (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen inactivation. Upon energy starvation, conditions in which 'respiratory protection' is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the 'iron-only' alternative nitrogenases exhibited similar growth rates irrespective of the presence or absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby 'respiratory protection' is the major physiological mechanism responsible for the protection of all three nitrogenases during energy-supplemented growth. Upon energy starvation, however, 'conformational protection', mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.
有人提出，棕色固氮菌的FeSII蛋白可介导钼依赖型固氮酶组分的“构象保护”，使其免受氧灭活。我们已克隆并鉴定了FeSII蛋白的结构基因（fesII基因座）。杂交研究表明，除了褐球固氮菌外，在许多经过充分研究的固氮细菌的不同物种中均未发现fesII样基因。fesII基因座在固氮和非固氮条件下均有转录表达，尽管其转录本水平在固氮过程中上调了约2.5倍。通过引物延伸分析确定了启动子区域，其与其他σ70型启动子相似。构建了缺失FeSII蛋白的突变体。这些突变体在非固氮和固氮生长过程中，在多种碳源上的生长特性与野生型菌株基本无差异。然而，与野生型相比，突变体细胞无细胞提取物中的固氮酶活性对不可逆氧灭活更为敏感。用250 mM NaCl处理（已知该条件可使FeSII与固氮酶组分解离）时，野生型和突变体提取物对氧灭活同样敏感。在能量饥饿（“呼吸保护”不起作用的条件）下，与野生型相比，缺失突变体中固氮酶的钼铁蛋白和铁蛋白在体内降解速度要快得多。依赖钒或“仅铁”替代固氮酶的菌株，无论是否存在FeSII蛋白，生长速率相似，并且钒固氮酶组分的体外失活不受FeSII蛋白缺失的影响。总而言之，这些结果与一个模型一致，即“呼吸保护”是能量充足生长期间保护所有三种固氮酶的主要生理机制。然而，在能量饥饿时，由FeSII蛋白介导的“构象保护”能够暂时保护传统的钼固氮酶组分免受过失活和随后的降解。
