ROLE OF THE HAEMOPHILUS-DUCREYI TON SYSTEM IN INTERNALIZATION OF HEMEFROM HEMOGLOBIN

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 h gbA 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 o nly at high concentrations of heme, was TonB independent, and demonstr ated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wi ld-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to util ize hemoglobin but able to utilize hemin at the same levels as the par ent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests t hat uptake of hemin in H. ducreyi is TonB independent. Additionally, t he H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with dere pression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes reve aled that the arrangement of the genes was exbB exbD tonB; The proximi ty and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino aci d sequences Of these H. ducreyi genes, was most similar to those from other pasteurellae.