Elkins C, Totten P A, Olsen B, Thomas C E
Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill 27599, USA.
Infect Immun. 1998 Jan;66(1):151-60. doi: 10.1128/IAI.66.1.151-160.1998.
By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressing hgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes was exbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.
通过克隆到大肠杆菌中并构建杜氏嗜血杆菌的同基因突变体，我们发现血红蛋白受体（HgbA）依赖于托蛋白（TonB）。表达杜氏嗜血杆菌hgbA的大肠杆菌hemA tonB突变体仅在存在完整的杜氏嗜血杆菌Ton系统质粒时，才能在低水平血红蛋白作为血红素来源的培养基上生长。相比之下，表达hgbA的大肠杆菌hemA tonB突变体在高浓度血红素条件下才能利用血红素生长，且不依赖于TonB，这表明杜氏嗜血杆菌HgbA不足以在大肠杆菌中作为典型的依赖TonB的血红素受体发挥作用。用exbB、exbD和tonB缺失突变体替换野生型杜氏嗜血杆菌的exbB、exbD和tonB基因座，得到一个杜氏嗜血杆菌同基因突变体，该突变体无法利用血红蛋白，但能够以与亲本菌株相同的水平利用血红素以满足其血红素需求。这一发现证实了HgbA介导的血红蛋白利用对TonB的依赖性，并表明杜氏嗜血杆菌中血红素的摄取不依赖于TonB。此外，杜氏嗜血杆菌Ton系统突变体合成了更多的HgbA和其他血红素调节的外膜蛋白，这与突变体中由于细胞内血红素和/或铁浓度降低导致这些蛋白的去阻遏作用一致。对Ton系统基因的测序表明，这些基因的排列顺序是exbB exbD tonB。这些基因的邻近性和结构表明它们作为一个操纵子进行转录。这种排列方式，以及这些杜氏嗜血杆菌基因的DNA和推导的氨基酸序列，与其他巴斯德氏菌的最为相似。
