Bowen S W, Hassan H M
Department of Biochemistry, North Carolina State University, Raleigh 27695.
Arch Biochem Biophys. 1993 May;302(2):372-9. doi: 10.1006/abbi.1993.1226.
The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell. To further investigate the mode of regulation of the gene (sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied. The mutant strains expressed beta-galactosidase under anaerobic conditions, whereas the wild-type did not. Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2'-dipyridyl, or to the redox cycling compound, paraquat. The wild-type, however, did respond to these effectors. In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5). Replacement of the wild-type 5'-regulatory region with either of the mutants' cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase. Sequence and restriction analysis revealed the presence of an IS2 insertion element in the promoter region of one of the mutants (NC5). This insertion caused the displacement of the 5'-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident-10 region from sodA and -35 from IS2. The second mutation (from NC4) was similarly analyzed, and an IS5 element was identified. The insertion site of IS5 (in NC4) was 6 bp (5'-TTAATT-3') upstream from the IS2 site (in NC5). Anaerobic expression of sodA in NC4 was lower than in NC5. This difference was almost eliminated in an arc- background, suggesting that the sequence 5'-TTAATT-3' might be essential for negative regulation by ArcA.
含锰超氧化物歧化酶的生物合成受影响细胞氧化还原电位的刺激调控。为进一步研究编码该酶的基因（sodA）的调控模式，对含有sodA::lacZ基因融合的菌株中的顺式作用调控突变进行了研究。突变菌株在厌氧条件下表达β-半乳糖苷酶，而野生型则不表达。此外，突变体不会因铁螯合剂2,2'-联吡啶或氧化还原循环化合物百草枯的存在而被诱导。然而，野生型对这些效应物有反应。体内克隆用于从突变体（NC4和NC5）中分离顺式作用调控元件。用任何一个突变体的顺式作用调控元件替换野生型5'-调控区，都会导致活性锰超氧化物歧化酶的厌氧表达。序列和限制性分析显示其中一个突变体（NC5）的启动子区域存在一个IS2插入元件。这种插入导致sodA的5'-调控区移位，并形成一个由sodA的常驻-10区和IS2的-35区组成的功能性杂合启动子。对第二个突变体（来自NC4）进行了类似分析，鉴定出一个IS5元件。IS5（在NC4中）的插入位点在IS2位点（在NC5中）上游6 bp（5'-TTAATT-3'）处。NC4中sodA的厌氧表达低于NC5。在arc-背景下，这种差异几乎消除，表明序列5'-TTAATT-3'可能对ArcA的负调控至关重要。
