Table 1

The L. pneumophila dot virulence loci are required to transfer RSF1010 plasmid into recipient bacteria. Mating was assayed by mixing 1.0 × 109 L. pneumophila containing noted plasmids with a tenfold excess of a recipient bacteria strain. Matings were performed in triplicate by allowing the mixed bacterial cultures to incubate for 2 hours at 37°C on 45-mm Millipore hemagglutinin filters (HAWP 047 S0) placed onto prewarmed charcoal-yeast extract media with thymidine (CYET) plates (3).

Donor strain*MediaRecipientNumber of conjugants per donor§
RSF1010CYETLp013.9 × 10−6
RSF1010CYETE. coli ER17936.6 × 10−7
RSF1010CYETE. coliMM2942.2 × 10−7
RSF1010CYET + DNase ILp013.2 × 10−6
RSF1010CYET + DNase IE. coliER17939.1 × 10−7
RSF1010CYET + DNase IE. coli MM2942.4 × 10−7
RSF1010 ΔoriT CYETLp01< 3.8 × 10−9
RSF1010 ΔoriT CYETE. coliER1793< 4.0 × 10−9
RSF1010 ΔoriT CYETE. coli MM294< 3.8 × 10−9
RSF1010CYETE. coliC600< 3.7 × 10−9
Donor strain|Donor plasmidRecipientNumber of conjugants per donor#
Wild typepKB5E. coliMM2942.5 × 10−6
dotG pKB5E. coliMM294< 4.3 × 10−9
dotG pdotG + E. coliMM2943.5 × 10−6
dotB pKB5E. coliMM294< 5.6 × 10−9
dotB pdotB + E. coliMM2944.8 × 10−6
dotA pKB5E. coli MM294< 6.1 × 10−9
dotA pdotA + E. coliMM2949.2 × 10−6
icmWXYZ pKB5E. coli MM294< 1.0 × 10−8
icmWXYZ picmWXYZ + E. coli MM2943.4 × 10−6
dotE pKB5E. coliMM294< 5.8 × 10−9
dotE pdotE + E. coliMM2948.7 × 10−6
  • * Lp02, a replication-competent strain (3), was transformed by electroporation with either RSF1010 Kan, an RSF1010 plasmid containing kanamycin from Tn903 (RSF1010 in table) (18), or RSF1010 ΔoriT, an RSF1010 plasmid containing a deletion (Δ13) in the origin of transfer (oriT) that completely abolishes conjugation (18).

  • Matings were performed on CYET or CYET containing DNase I (1 μg/ml).

  • Recipients were either a L. pneumophila strain competent for intracellular growth (Lp01) (3), the restriction minus E. coli strains ER1793 (hsdR) and MM294 (hsdR), or the restriction-competent E. coli strain C600 (19).

  • § Legionella pneumophilaconjugants were selected on charcoal-yeast extract lacking thymidine to select against the thymine auxotrophic donor Lp02, as well as kanamycin at 20 μg/ml to select for plasmid transfer. Escherichia coli conjugants were selected on LB plates containing kanamycin at 25 μg/ml. The L. pneumophila donor strains are unable to grow on LB plates.

  • | The donor strains are Lp02 (wild type), the dotG deletion strain (JV573), and the following four salt-resistant mutants (6): JV303 (dotB), JV309 (dotA), JV312 (icmWXYZ), and JV328 (dotE).

  • The donor plasmids were either the vector pKB5, an RSF1010 plasmid harboring AmpR(3), or pKB5 containing the complementing ORFs for the various mutants.

  • # Escherichia coli conjugants were selected on LB plates containing ampicillin (150 μg/ml). Reversion rates of markers used to select conjugants were substantially below the rates of transfer detected (for example, Lp02 + RSF1010 donor, < 9.1 × 10−11; Lp01 as a recipient, < 7.7 × 10−11; E. coli ER1793 as a recipient, < 1.6 × 10−11; and E. coliMM294 as a recipient, < 1.1 × 10−10).