Graeff-Wohlleben H, Killat S, Banemann A, Guiso N, Gross R
Lehrstuhl fur Mikrobiologie, Biozentrum, Universität Wurzburg, Germany.
J Bacteriol. 1997 Apr;179(7):2194-201. doi: 10.1128/jb.179.7.2194-2201.1997.
The Fur titration assay (FURTA) recently developed by I. Stojiljkovic and coworkers (J. Mol. Biol. 236:531-545, 1994) was applied to clone iron-regulated genes of Bordetella pertussis. After sequence analysis, one of the clones obtained by this selection procedure was shown to contain an open reading frame with significant sequence similarities to Mn-containing superoxide dismutases (SodA). The open reading frame was preceded by a Fur consensus binding site, which according to primer extension analysis overlaps the -10 region of the sodA promoter. Southern blot analysis also revealed the presence of sodA homologous sequences in Bordetella bronchiseptica. On the transcriptional level, sodA expression is strictly iron regulated in both organisms and also in the heterologous host Escherichia coli harboring a plasmid with the sodA gene. Accordingly, SodA-mediated superoxide dismutase activity in Bordetella lysates was detected only after cultivation of the bacteria in iron-restricted media. A B. bronchiseptica fur mutant constitutively expressed SodA, thereby confirming the functional similarity of the iron regulatory systems in the two genera. Apart from iron regulation, sodA expression was affected by changes in DNA topology induced by coumermycin A but not by the global virulence regulatory Bvg system. B. pertussis and B. bronchiseptica sodA deletion mutants did not show significant changes in their growth properties. In contrast, mutation of the previously described Fe-containing SodB enzyme resulted in clones strongly impaired in viability. No direct involvement of SodA in bacterial virulence could be revealed because deletion of the sodA gene affected survival of Bordetella species neither in cultured macrophages nor in a mouse respiratory infection model.
I. Stojiljkovic及其同事最近开发的Fur滴定分析方法（FURTA）（《分子生物学杂志》236:531 - 545，1994年）被用于克隆百日咳博德特氏菌的铁调节基因。经过序列分析，通过该筛选程序获得的一个克隆显示含有一个开放阅读框，与含锰超氧化物歧化酶（SodA）具有显著的序列相似性。该开放阅读框之前有一个Fur共有结合位点，根据引物延伸分析，该位点与sodA启动子的 - 10区域重叠。Southern印迹分析还揭示了支气管败血博德特氏菌中存在sodA同源序列。在转录水平上，sodA在这两种细菌以及携带含有sodA基因质粒的异源宿主大肠杆菌中均受到严格的铁调节。因此，只有在铁限制培养基中培养细菌后，才能在百日咳博德特氏菌裂解物中检测到SodA介导的超氧化物歧化酶活性。支气管败血博德特氏菌的fur突变体组成型表达SodA，从而证实了这两个属中铁调节系统的功能相似性。除了铁调节外，sodA的表达受香豆霉素A诱导的DNA拓扑结构变化的影响，但不受全局毒力调节Bvg系统的影响。百日咳博德特氏菌和支气管败血博德特氏菌的sodA缺失突变体在生长特性上没有显示出显著变化。相比之下，先前描述的含铁SodB酶的突变导致克隆的活力严重受损。由于sodA基因的缺失既不影响百日咳博德特氏菌在培养的巨噬细胞中的存活，也不影响其在小鼠呼吸道感染模型中的存活，因此未发现SodA直接参与细菌毒力。
