Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili

Abstract

We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).

FIG. 1

Transformation of L. pneumophila with plasmid DNA. Transformation frequency was measured after addition, at 4 μg/ml, of plasmid DNA from pUC4K, pBC-K, pBJ114 (pilE_L::Kan^r), pBJ115 (pilE_L::Kan^r), pJA2A-K2 (ppa::Kan^r), pGS-K (gspA::Kan^R), or pOHBOC1-K (asd::Kan^r) to L. pneumophila AA100. The number of chloramphenicol-resistant transformants was measured after addition of pBOC20. Loss of auxotrophy for DAP was measured after addition of 4 μg of pOHBOC1 or pOHBOC2 plasmid DNA per ml to L. pneumophila AA400. The limit of detection for transformation frequency was 10⁻⁹. Error bars indicate standard deviations.

FIG. 2

DNA concentration dependence of transformation with plasmid DNA. Transformation frequency was measured after addition of the indicated amount of plasmid DNA. The limit of detection for transformation frequency was 10⁻⁹. Error bars indicate standard deviations.

FIG. 3

Position of the Kan^r marker in cloned DNA fragments. The relative position of the marker is shown for pBJ114 and pBJ115 (pilE), pOHBOC1-K (asd), pGS-K (gspA), and pJA2A-K2 (ppa). A 1-kb fragment is shown for scale.

FIG. 4

Induction of competence in L. pneumophila. (Top) Viable counts before addition of DNA. Sample numbers correspond to samples taken at the time points indicated in the bottom panel. (Bottom) L. pneumophila BS200 chromosomal DNA (400 μg/ml) was added to cultures at the time points indicated, and the cultures were allowed to incubate for an additional 2 days before transformation frequency was determined. No transformants were detected after addition of DNA at days 0 and 6. The limit of detection for transformation frequency was 10⁻⁹. Error bars indicate standard deviations.