Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome

Abstract

Transposon TnS was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by coijugal transfer of the plasmid to Escherichia coli. TnS insertions were obtaied by culturing L. pneumophia at the nonpermissive temperature (43°C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked TnS-containing strains, and Southern hybridizations indicated that TnS, but not pRK340, inserted into multiple sites in the Legioneila chromosome. In addition, the streptomycin resistance determinant on TnS was expressed in L. pneumophila.Legionella pneumophila and related species are fastidious facultative intracellular parasites for which determinants of virulence have not been clearly established. Continued propagation of virulent strains on laboratory media leads to a loss of virulence (15), which can be restored by passage of the avirulent strains in cell culture (24). Despite transitions between virulence and avirulence, no phenotypic markers have been found which correlate with loss of virulence. Although plasmid-encoded virulence factors have been reported for other gram-negative bacteria (18,19), no correlation between plasmids and virulence has been made for the legionellae (1, 5). Genetic regulation of the transition from virulence to avirulence in the legionellae may be under control mechanisms similar to those recently described for Bordetella pertussis (23). Weiss and Falkow (23) demonstrated that the phase change (reversible conversion from virulence to avirulence) of B. pertussis was controlled by a chromosomal trans-acting gene product. However, before progress can be made towards resolving mechanisms of pathogenesis for the legionellae, it will be necessary both to develop a workable genetic system and to identify specific markers associated with virulence.Since transposon mutagenesis has proved to be an effective tool in studies of pathogenesis, we set out to develop a means for delivering transposons into the chromosome of L. pneumophila. It has recently been reported that Pseudomonas plasmids of the IncP-1 incompatibility group can be transferred to several Legionella species via conjugation (8). Chen et al. (6) have introduced TnS into several Legionella species on the broad-host-range suicide plasmid pJB4JI, but they did not demonstrate transposition. In this study we show that pRK340, a temperature-sensitive derivative of the IncP-1 plasmid RK2, can effectively deliver Tn5 to the chromosome of L. pneumophila.All strains and plasmids used in this study are listed in Table 1. L. pneumophila strains were maintained on ACES (N-(2-acetamido)-2-aminoethanesulfonic acid)-buffered charcoal-yeast extract (BCYE) agar (17), and batch cultures were grown in BYE medium (without charcoal) and harvested as previously described (12). All cultu...

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

“…Heterospecific matings were carried cut between Escherichia coli harbouring Tn5tac1 and LpO2, selecting for kanamycin-and streptomycin resistance. In actual experiments, transposition of the Tn5 derivative did net occur at high frequency, consistent with a previous study (Keen et at., 1985). To enrich for L. pneumophita intracellular growth mutants, independent pools of exccnjugant bacteria were subjected to three cycles of intracellular thymineless death enrichment.…”

Genetic approaches to study Legionella pneumophila pathogenicity

“…For complementation, stable replicating plasmids, carrying the gene under question were transferred to the mutant. Broad host range plasmids of the incompatibility groups incl' and incQ have been shown to be useful for this purpose [48,55,60,81]. Efficient transfer occurs by mobilization of the plasmids with a mob site by chromosomally encoded Tra functions or by helper plasmids, so that the size of the transferred plasmids is small, increasing the rate of transfer.…”

“…Another important point in genetic transfer is the counterselection on the incoming DNA. For this purpose many authors have used the kanamycin resistance gene which is well expressed in L. pneurnophila [17,31,48,89]. 3"he chloramphenicol acetyl tranferase seems to be poorly expressed in L. pneumophila [60,811, so that the antibiotic concentration is crucial.…”

“…2). Keen et al [48] were the first to report on Tn5 mutagenesis using a derivative of an incP ts-plasmid. In the mean- Arroyo ct al.…”

Random mutagenesis of Legionella pneumophila with mini-Tn10

“…Thus far, three transposable elements, Tn5, Mu, and Tn903, have been used to mutagenize L. pneumophila. Unfortunately, Tn5 transposes at very low frequency in Legionella and may excise when its host is passaged without antibiotic selection [1,2,3]. Although * Corresponding author.…”