Cornelissen C N, Biswas G D, Tsai J, Paruchuri D K, Thompson S A, Sparling P F
Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill 27599.
J Bacteriol. 1992 Sep;174(18):5788-97. doi: 10.1128/jb.174.18.5788-5797.1992.
The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.
致病性奈瑟菌能够将转铁蛋白作为其唯一的铁源。一种奈瑟菌转铁蛋白受体已被鉴定并明确了其特性；然而，转铁蛋白受体功能所需蛋白质的生化特性尚未确定。我们在淋病奈瑟菌中鉴定出两种铁抑制性转铁蛋白结合蛋白，即TBP1和TBP2。我们采用了两种方法来克隆淋病奈瑟菌转铁蛋白受体功能所需的基因。首先，用针对TBP1产生的多克隆抗血清来鉴定表达TBP1表位的克隆。其次，克隆了一个野生型基因拷贝，该拷贝修复了转铁蛋白受体功能（trf）突变体中的缺陷。通过这两种方法获得的克隆经DNA测序显示相互重叠。对这两个克隆进行转座子诱变，并将诱变片段重组到淋病奈瑟菌染色体中，产生了一些突变体，这些突变体显示转铁蛋白与全细胞的结合减少，并且无法在转铁蛋白上生长。在这些突变体中没有产生TBP1，但TBP2的表达正常。编码淋病奈瑟菌TBP1（tbpA）的基因的DNA序列预测了一个与大肠杆菌和恶臭假单胞菌的TonB依赖性外膜受体同源的蛋白质序列。因此，TBP1的功能和预测的蛋白质序列都与该蛋白作为转铁蛋白受体的功能一致。
