The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element.
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.
Viswanathan
V K
VK
Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Kurtz
Sherry
S
Pedersen
Lisa L
LL
Abu-Kwaik
Yousef
Y
Krcmarik
Kevin
K
Mody
Sejal
S
Cianciotto
Nicholas P
NP
D000048:Acanthamoeba / Q000382:microbiology; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D005816:Genetic Complementation Test; D007501:Iron / Q000378:metabolism*; D016952:Legionella pneumophila / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D008565:Membrane Proteins / Q000235:genetics*; D016366:Open Reading Frames; D013045:Species Specificity; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C116246:CcmC protein, bacteria; D004251:DNA Transposable Elements; D008565:Membrane Proteins; D007501:Iron