Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.

Pairs of primers flanking known miniTn10 transposon insertion sites were used to confirm the presence of the transposon in DNA isolated from Legionella pneumophila mutants. It was expected that the polymerase chain reaction products derived from the mutant template would be larger than those from the wild-type (WT) template due to the presence of the 1.8-kb transposon. Instead, it was observed that the mutant template yielded a product of almost the same size as that yielded by WT template. We present evidence to indicate that the aberrant product from the mutant template is a direct result of secondary structure of the template resulting from an inverted repeat sequence present in the miniTn10 transposon.

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