Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene

Abstract

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 during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype 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 fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.