B-282. Characterization of a Putative Heme ABC Transporter in Neisseria meningitidis

K. T. Ingrey, B. C. Lee;
Univ. of Ottawa, Ottawa, ON, CANADA.

A detailed mechanism for heme uptake in Neisseria meningitidis has not yet been elucidated. Once heme is deposited in the periplasmic space, a heme dedicated ABC transporter has been hypothesized to convey heme into the cytoplasm. We have previously identified an ABC transporter engaged in heme transport in Haemophilus ducreyi. A BLAST search revealed an uncharacterized meningococcal protein exhibiting 27% amino acid identity to the periplasmic-binding protein in the H. ducreyi transporter. The gene encoding this putative periplasmic binding protein is located downstream of two open reading frames organized in a putative operon displaying sequences indicative of an ABC transporter. Prior studies have shown enhanced expression of this neisserial protein under low iron conditions leading us to hypothesize that this ABC transporter is involved in meningococcal heme acquisition. As the initial step in addressing this hypothesis, we constructed a mutant in the periplasmic-binding protein gene by insertional inactivation with a selectable antibiotic marker. The wild-type periplasmic gene was cloned by PCR amplification from a clinical meningococcal N. meninigitidis serogroup B strain Barden using site-specific primers encompassing the entire coding region. The gene was disrupted with the insertion of a chloramphenicol cassette containing a Neisseriae uptake sequence and a transcriptional terminator. Following transformation of the parental strain with the linearized plasmid construct, proper allelic exchange was confirmed by PCR. The ability of various iron sources (2 μM and 5 μM heme, 0.5 μM hemoglobin and 5 μM FeCl3) to support the growth of the mutant and wild-type strains was compared under iron-restrictive conditions. No difference in growth rates between the two strains was discerned. Although the mutant failed to display the predicted heme-deficient phenotype, the role of this operon in meningococcal heme uptake requires further exploration, in view of the recognized degenerate binding specificity of the permease component of some ABC transporters. Such mutation analysis of the permease gene is underway.