Fig 4.

Relative quantification of the three episomal forms of LpcGI-2 and conjugation frequency. (A) SYBR green quantitative PCR was done with chromosomal DNA from L. pneumophila wild-type, Δlpc1833, Δlpc1884, and Δlpc2123 strain in exponential (E) and stationary (S) growth phases. Circularization of LpcGI-2 was detected with primers RT-LpcGI-2-2R/RT-LpcGI-2-3U for GI-2-A, RT-LpcGI-2-1U/RT-LpcGI-2-5R for GI-2-B, and RT-LpcGI-2-3U/RT-LpcGI-2-5R for GI-2-AB. The flaA gene served as a chromosomal control, and the relative amount of copies was calculated in relation to a standard curve. Results are means of three independent experiments. Statistical significance is characterized by symbols above the columns: ∞, comparison of GI-2-B and GI-2-AB versus GI-2-A of the wild type; ∼, comparison of the episomal forms of the mutant strains versus the wild type. (B) For conjugation experiments, L. pneumophila Corby wild type (WT*) or the Δlpc1833 and ΔpilT mutant strains were used as the donor and the L. pneumophila Philadelphia I JR32 strain served as the acceptor. Conjugation was done at 30°C on BCYE agar plates in the presence of DNase I. The transconjugation rates (ratios of transconjugants/donor) were 7.3 × 10⁻³ for the wild-type strain, 5 × 10⁻⁵ for the Δlpc1833 mutant strain, and 4.9 × 10⁻⁵ for the ΔpilT mutant strain. Results of the conjugation experiments are means of two independent experiments.