Deneer H G, Boychuk I
Department of Microbiology, University of Saskatchewan, Saskatoon, Canada.
Can J Microbiol. 1993 May;39(5):480-5. doi: 10.1139/m93-068.
One mechanism by which Listeria monocytogenes is thought to obtain iron required for growth is through the extracellular reduction of a ferric iron source to the ferrous form. To better characterize this reductase activity we have developed a simple plate assay that allows detection of colonies of Listeria species able to reduce ferric iron. Cells are plated on an agar base medium containing a ferric iron source and ethylenediamine dihydroxyphenylacetic acid. Colonies are then overlain with soft agarose containing NADH, flavin mononucleotide, and Ferrozine, a chelator of ferrous iron. Colonies able to reduce the ferric iron source form a red-purple color as the ferrous iron is complexed with ferrozine. Using this qualitative assay we have shown that all species of Listeria are able to reduce ferric iron when presented as ferric ammonium citrate whereas most other species of Gram-positive and Gram-negative bacteria are not. Only Clostridium perfringens was able to reduce ferric iron to the same extent as Listeria. Listeria monocytogenes was further shown to be capable of reducing various ferric iron salts as well as iron bound to ferritin, transferrin, and 2,3-dihydroxybenzoic acid in the agar plate assay. The utility of this assay was demonstrated by using it to screen a bank of Tn916-derived mutants of L. monocytogenes for clones unable to reduce ferric iron. Four such mutants were identified and all were shown to have greatly decreased ferric reductase activity.
单核细胞增生李斯特菌被认为获取生长所需铁的一种机制是通过将三价铁源细胞外还原为二价铁形式。为了更好地表征这种还原酶活性，我们开发了一种简单的平板试验，可检测能够还原三价铁的李斯特菌属菌落。将细胞接种在含有三价铁源和乙二胺二羟基苯乙酸的琼脂基础培养基上。然后在菌落上覆盖含有NADH、黄素单核苷酸和亚铁嗪（一种二价铁螯合剂）的软琼脂糖。能够还原三价铁源的菌落会形成红紫色，因为二价铁与亚铁嗪络合。使用这种定性试验，我们发现当以柠檬酸铁铵形式存在时，所有李斯特菌属都能够还原三价铁，而大多数其他革兰氏阳性和革兰氏阴性细菌则不能。只有产气荚膜梭菌能够与李斯特菌一样程度地还原三价铁。在琼脂平板试验中，进一步证明单核细胞增生李斯特菌能够还原各种三价铁盐以及与铁蛋白、转铁蛋白和2,3 - 二羟基苯甲酸结合的铁。通过使用该试验筛选一组来源于Tn916的单核细胞增生李斯特菌突变体，以寻找不能还原三价铁的克隆，证明了该试验的实用性。鉴定出四个这样的突变体，并且所有突变体均显示出三价铁还原酶活性大大降低。
