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SirR, a Novel Iron-Dependent Repressor inStaphylococcus epidermidis

机译：SirR，一种新型的铁依赖性表皮葡萄球菌阻遏物

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In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation. To gain insights into the molecular basis of iron-dependent gene regulation in the staphylococci, we sequenced the DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to iron stress, for example, production of diphtheria toxin, siderophore, and a heme oxygenase. Upstream of thesitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a polypeptide of approximately 25 kDa with homology to the DtxR family of metal-dependent repressor proteins. This ORF has been designated SirR (staphylococcal iron regulator repressor). Within thesitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start ofsitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR protein was overexpressed, purified, and used in DNA mobility shift assays; SirR retarded the migration of a synthetic oligonucleotide based on the Sir box in a metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus,S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent metal cation-dependent transcriptional repressor which is widespread among the staphylococci.
机译：在表皮葡萄球菌和金黄色葡萄球菌中，许多细胞壁和细胞质膜相关的脂蛋白响应铁饥饿而被诱导。为了深入了解葡萄球菌中铁依赖性基因调控的分子基础，我们对3-kb S上游的DNA进行了测序。 Northern blot分析表明，表皮中的sitABC 操纵子受生长培养基中铁含量的转录调控。我们鉴定了两个与白喉棒状杆菌DtxR regulon元件同源的DNA序列，该铁质响应铁胁迫控制例如白喉毒素，铁载体和血红素加氧酶的产生。 sitABC 操纵子的上游并有不同转录的是一个645 bp的开放阅读框（ORF），其编码约25 kDa的多肽，与金属依赖性阻遏蛋白的DtxR家族同源。该ORF被指定为SirR（葡萄球菌铁调节物阻遏物）。在 sitABC 启动子/操纵子区域内，我们还发现了一个与 sitABC 转录起始重叠的dyad对称区域，该区域与DtxR操纵子共有序列具有高度同源性，这表明称为Sir盒的区域是SirR结合位点。 SirR蛋白被过表达，纯化并用于DNA迁移率变动分析; SirR抑制了基于Sir盒的合成寡核苷酸在金属（Fe 2 + 或Mn 2 + ）依赖性方式中的迁移，提供了证实性证据表明该基序是SirR结合位点。此外，用合成的Sir盒作为探针对葡萄球菌染色体DNA的Southern印迹分析证实 S中至少有五个Sir盒。表皮基因和 S基因组中的至少三个。金黄色葡萄球菌，表明SirR控制着多个靶基因的表达。使用针对SirR的单特异性多克隆抗体探查 S全细胞裂解物的Western印迹。金黄色， S。肉食性动物， S。表皮， S。 Hominis ， S。 cohnii ， S。 lugdunensis 和 S。溶血性，我们在所检测的每个葡萄球菌物种中鉴定出大约25 kDa的交叉反应蛋白。综上所述，这些数据表明，SirR起着依赖于二价金属阳离子的转录阻遏物的作用，在葡萄球菌中广泛存在。

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