Butler Catherine A, Dashper Stuart G, Zhang Lianyi, Seers Christine A, Mitchell Helen L, Catmull Deanne V, Glew Michelle D, Heath Jacqueline E, Tan Yan, Khan Hasnah S G, Reynolds Eric C
Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Victoria, Australia.
PLoS One. 2014 Nov 6;9(11):e111168. doi: 10.1371/journal.pone.0111168. eCollection 2014.
Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.
牙龈卟啉单胞菌是一种革兰氏阴性病原体，与生物膜介导的慢性牙周炎疾病相关。牙龈卟啉单胞菌生物膜的形成依赖于环境血红素，由于其无法从头合成原卟啉IX，因此对血红素有绝对需求，所以牙龈卟啉单胞菌会转运从人类宿主中释放的铁和血红素。在革兰氏阴性细菌中，多种过渡金属离子的稳态通常在转录水平上由铁摄取调节因子（Fur）超家族的成员介导。牙龈卟啉单胞菌有一个预测的Fur超家族直系同源物，我们将其命名为Har（血红素相关调节因子）。重组Har在Zn2+存在下形成二聚体，每个单体以高亲和力（解离常数为0.23μM）结合一个血红素分子。血红素的结合导致Zn（II）Har的构象变化，97位半胱氨酸残基作为预测的-97C-98P-99L-血红素结合基序的一部分参与血红素结合。与野生型相比，在Har突变体（ECR455）中35个基因的表达下调，9个基因上调。先前发现26个下调基因在作为生物膜生长的牙龈卟啉单胞菌中上调，11个基因在血红素限制条件下上调。截短的Zn（II）Har结合了ECR455突变体中上调基因之一dnaA（PGN_0001）的启动子区域。随着血红素浓度的增加，这种结合减少，这与基因表达受血红素可用性调节一致。ECR455形成的生物膜明显少于野生型，并且与野生型不同，生物膜的形成与血红素可用性无关。牙龈卟啉单胞菌拥有一个血红素结合的Fur直系同源物，可调节血红素依赖性生物膜的形成。
