Lee J H, Wang T, Ault K, Liu J, Schmitt M P, Holmes R K
Department of Microbiology, University of Colorado Health Sciences Center, Denver 80262, USA.
Infect Immun. 1997 Oct;65(10):4273-80. doi: 10.1128/iai.65.10.4273-4280.1997.
DtxR is a dimeric, sequence-specific, DNA-binding protein that functions as an iron-dependent, negative global regulator in Corynebacterium diphtheriae. Under high-iron conditions, DtxR represses the synthesis of diphtheria toxin, corynebacterial siderophore, and other components of the high-affinity iron uptake system. Three DtxR-regulated promoter/operators designated tox, IRP1, and IRP2 were reported previously. In this study, we identified and characterized three additional DtxR-regulated promoter/operators from C. diphtheriae designated IRP3, IRP4, and IRP5. When beta-galactosidase was expressed from these three new promoter/ operators in Escherichia coli containing dtxR+ on pDSK29, enzyme levels were 5- to 30-fold lower during high-iron growth than during low-iron growth. In gel shift assays, the mobility of DNA fragments containing each promoter/operator decreased in the presence of purified DtxR and Co2+. In footprinting assays, DtxR protected 36-, 35-, and 30-bp regions of IRP3, IRP4, and IRP5, respectively, from cleavage by DNase I. In the 19-bp core of each promoter/operator, 12 or 13 bp matched the consensus for the DtxR-binding site. The putative polypeptides encoded by the open reading frames (ORFs) downstream from IRP3 and IRP4 were homologous, respectively, to several bacterial transcriptional regulators and to the deduced polypeptide encoded by an ORF located between the E. coli genes for primosomal replication protein N and adenine phosphoribosyltransferase. The putative polypeptide encoded by the ORF downstream from IRP5 was not homologous to any sequence in the protein database at the National Center for Biotechnology Information. When the ORFs downstream from IRP3 and IRP4 were expressed under the control of the phage T7 promoter in E. coli, polypeptide products of the predicted sizes were detected in small amounts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
DtxR是一种二聚体、序列特异性的DNA结合蛋白，在白喉棒状杆菌中作为铁依赖性的负向全局调节因子发挥作用。在高铁条件下，DtxR抑制白喉毒素、棒状杆菌铁载体以及高亲和力铁摄取系统其他成分的合成。先前已报道了三个由DtxR调节的启动子/操纵子，分别命名为tox、IRP1和IRP2。在本研究中，我们从白喉棒状杆菌中鉴定并表征了另外三个由DtxR调节的启动子/操纵子，命名为IRP3、IRP4和IRP5。当在含有pDSK29上的dtxR+的大肠杆菌中从这三个新的启动子/操纵子表达β-半乳糖苷酶时，高铁生长期间的酶水平比低铁生长期间低5至30倍。在凝胶迁移试验中，含有每个启动子/操纵子的DNA片段在纯化的DtxR和Co2+存在下迁移率降低。在足迹试验中，DtxR分别保护IRP3、IRP4和IRP5的36bp、35bp和30bp区域不被DNase I切割。在每个启动子/操纵子的19bp核心区域中，12或13bp与DtxR结合位点的共有序列匹配。IRP3和IRP4下游开放阅读框（ORF）编码的推定多肽分别与几种细菌转录调节因子以及位于大肠杆菌基因间的ORF编码的推定多肽同源，该ORF位于引发体复制蛋白N和腺嘌呤磷酸核糖转移酶基因之间。IRP5下游ORF编码的推定多肽与美国国立生物技术信息中心蛋白质数据库中的任何序列均无同源性。当IRP3和IRP4下游的ORF在噬菌体T7启动子的控制下在大肠杆菌中表达时，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测到少量预测大小的多肽产物。
