ESCHERICHIA-COLI F PLASMID TRANSFERS TO AND REPLICATES WITHIN LEGIONELLA-PNEUMOPHILA - AN ALTERNATIVE TO USING AN RP4-BASED SYSTEM FOR GENEDELIVERY

Derivatives of the self-transmissible F plasmid of Escherichia coli ca n be introduced into Legionella pneumophila by conjugation and maintai ned within only upon selection. In L. pneumophila, F-based replicons s eem to exist as extrachromosomal elements since they were readily lost when F-containing L. pneumophila was grown on nonselective medium. Th e F-based plasmids were not self-transmissible in L. pneumophila. The mating defect may be due to an inability to form the F pilus since F-c ontaining strains of L. pneumophila could neither be infected with the pilus-specific phage M13 nor transduced with fl-packaged ColEI replic ons. Currently, the most commonly used transfer system for introducing genetic information into L. pneumophila employs E. coli donors with a chromosomally integrated copy of RP4::Mu to mobilize plasmids bearing the RK2 origin of transfer (oriT). Use of this system to deliver Tnph oA for mutagenesis of the L. pneumophila chromosome led to transconjug ants that all contained cryptic DNA alterations that involved the plas mid RP4 and phage Mu. No TnphoA transposition was observed in L. pneum ophila. The fact that F-mediated conjugation can be used to efficientl y transfer plasmids containing the oriT of F to L. pneumophila provide s an important alternative to the RP4-based plasmid transfer system an d may avoid DNA anomalies in transconjugants that impede genetic analy sis. Furthermore, our results demonstrate the promiscuous nature of th e F conjugal transfer and replication systems. (C) 1994 Academic Press , Inc.