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An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases.

ABSTRACT: Legionella pneumophila, a parasite of alveolar macrophages, requires iron for intra- and extracellular growth. Although its mechanisms for iron assimilation are poorly understood, this bacterium produces Fur, a protein that can repress gene transcription in response to iron concentration. Because iron- and Fur-regulated genes are important for infection in other bacteria, the identification of similar genes in L. pneumophila was undertaken. A wild-type strain of L. pneumophila was randomly mutated with a mini-Tn10' lacZ transposon, and the resulting gene fusions were tested for iron regulation by assessing beta-galactosidase production in the presence and absence of iron chelators. Of the initial six mutants with iron-repressed lacZ fusions, two strains, NU229 and NU232, possessed fusions that were stably iron regulated. To assay for Fur regulation, the levels of beta-galactosidase were measured in strains no longer producing Fur. As in a number of pathogenic bacteria, L. pneumophila fur could not be insertionally inactivated, but spontaneous Fur- derivatives were generated by selecting for manganese resistance. Strain NU229 contained a Fur-repressed fusion based on derepression of lacZ expression in its manganese-resistant derivative. Extracellular growth of NU229 in bacteriological media was similar to that of wild-type strain 130b. To assess the role of an iron- and Fur-regulated (frgA) gene in intracellular infection, the ability of NU229 to grow within U937 cell monolayers was tested. Quantitative infection assays demonstrated that intracellular growth of NU229 was impaired as much as 80-fold. Reconstruction of the mutant by allelic exchange proved that the infectivity defect in NU229 was due to the inactivation of frgA and not to a second-site mutation. Subsequently, complementation of the interrupted gene by an intact plasmid-encoded gene demonstrated that the infectivity defect was due to the loss of frgA and not to a polar effect. Nucleotide sequence analysis revealed that the 63-kDa FrgA protein has homology with the aerobactin synthetases IucA and IucC of Escherichia coli, raising the possibility that L. pneumophila encodes a siderophore which is required for optimal intracellular replication. Southern hybridization analysis determined that frgA is specific to L. pneumophila.

SUBMITTER: Hickey EK

PROVIDER: S-EPMC174567 | biostudies-other | 1997 Jan

REPOSITORIES: biostudies-other

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An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases.

Hickey E K EK Cianciotto N P NP

Infection and immunity 19970101 1

Legionella pneumophila, a parasite of alveolar macrophages, requires iron for intra- and extracellular growth. Although its mechanisms for iron assimilation are poorly understood, this bacterium produces Fur, a protein that can repress gene transcription in response to iron concentration. Because iron- and Fur-regulated genes are important for infection in other bacteria, the identification of similar genes in L. pneumophila was undertaken. A wild-type strain of L. pneumophila was randomly mutat ...[more]

PMID: 8975903

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Project description: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.

Dataset Information

An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases.

Publications

An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases.

Similar Datasets

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