Iron acquisition in Listeria Monocytogenes

This investigation was designed to study iron acquisition mechanisms of Listeria monocytogenes. Molecular biology techniques were employed.;A listerial genomic library in E. coli and a library of listerial transposon mutants was constructed and screened. The assay used to screen both libraries was based on the ability of a ferrous iron chelator, ferrozine, to produce a red colour when complexed with ferrous iron. After screening approximately 10,000 clones from the genomic library and 12,000 mutants from the transposon library of mutants, 11 apparently ferric reductase-negative transposon mutants were identified. Subsequent analysis of these mutants showed them to be ferric reductase-positive. The reason for this reversal of phenotype is now known and was not investigated further.;To identify genes which were involved in the growth of L. monocytogenes in low-iron environments, the library of listerial transposon mutants were screened in low-iron medium. From the 5,500 screened, two mutants were identified which showed reduced ability to grow in both low-ferric and low-ferrous iron medium. In the high-iron medium, the growth of the mutants was similar to that of the wild type.;Mutant 9E was phenotypically similar to the wild type except for the inability to grow in low-iron medium. Mutant 7D had two additional differences; increased haemolytic activity and decreased motility when observed using phase microscopy. Sequencing of the flanking chromosomal DNA led to the identity of the genes which had been disrupted; flaA in mutant 7D and argiC in mutant 9E. Sequence analysis upstream from the flaA gene identified a new gene, cheV, in L. monocytogenes. The relationship of these mutations to the observed phenotypes was discussed. The ability of both mutants to grow in vivo was similar to that of the parental wild type.