MUTAGENESIS OF LEGIONELLA-PNEUMOPHILA USING TN903 DIILACZ - IDENTIFICATION OF A GROWTH-PHASE-REGULATED PIGMENTATION GENE

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression dur ing macrophage infection. A derivative of Tn903, Tn903dIIlacZ., is sho wn to transpose with high efficiency in L. pneumophila. Tn903dIIlacZ e ncodes resistance to kanamycin (Km(R)) and carries a 5' truncated 'lac Z gene that can form translational fusions to L. pneumophila genes upo n transposition. The cis-acting Tn903 transposase is supplied outside Tn903dIIlacZ, and hence chromosomally integrated copies are stable. Km (R) LacZ(+) insertion mutants of L. pneumophila were isolated and show n by DNA hybridization to carry a single Tn903dIIlacZ inserted within their chromosomes at various locations. One particular Km(R) LacZ(+) m utant, AB1156, does not produce the brown pigment(Pig(-)) characterist ic of Legionella species. Tn903dIIlacZ is responsible for this phenoty pe since reintroduction of the transposon-linked mutation into a wild- type background results in a Pig(-) phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig=lacZ f usion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig=lacZ expression also inc reased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill ho st macrophages.