Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob
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CLIFFORDS. MINTZ'AND CHANGH U A ZOU Department of Microbiology a n d Immunology, University of Miami, School of Medicine, P.O. Box 016960 (R-138), Miami, F L 33101, U.S.A. Received August 26, 1991 Revision received January 7, 1992 Accepted January 20, 1992 MINTZ, C. S., and Z o u , C . H. 1992. Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob. Can. J. Microbiol. 38: 664-671. RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionellapneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded per recipient for the markers tested. The presence of a Mu to recombinants at fequencies ranging from insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromoscme transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mucontaining donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila. Key words: Legionella pneumophila, chromosome transfer, Tn5-Mob, RK2::Mu. MINTZ, C. S., et Z o u , C. H. 1992. Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob. Can. J. Microbiol. 38 : 664-671. Des plasmides RK2::Mu et le transposon Tn5-Mob ont etk utilisks pour mobiliser le chromosome de Legionellapneumophila. Des conjugaisons en plaque entre les L. pneumophila donneurs contenant les plasmides RK2::Mu et les recepar receveur pour les marqueurs a veurs auxotrophes ont produit des recombinants a des frkquences de analyses. La presence d'une insertion Mu dans le chromosome des donneurs portant les plasmides RK2::Mu a augmentk la frequence du transfert chromosomique de certains marqueurs sklectionnes en comparaison avec les souches qui contenaient RK2::Mu seulement. Les experiences de cotransfert avec les donneurs contenant Mu et un auxotrophe pour la thymidine et le tryptophane n'ont pas permis de reveler un lien entre les locus thy et trp chez L. pneumophila. Une souche qui contenait une insertion chromosomique Tn5-Mob et le plasmide auxiliaire pRK24.4 a transfkrk les marqueurs chromosomiques a des frkquences de 10 - 7 par receveur. Ces rksultats sugghent que les plasmides RK2::Mu et le transposon Tn5-Mob peuvent 2tre utiles pour les experiences de cartographie genktique de L. pneumophila. Mots elks : Legionella pneumophila, transfert chromosomique, Tn5-Mob, RK2::Mu. [Traduit par la redaction]
Introduction Legionella pneumophila is a facultative intracellular pathogen capable of entering and growing in a variety of eukaryotic cells, including free-living amoebae (Fields et al. 1986), cultured animal cells (Dreyfus 1987; Oldham and Rodgers 1985), and alveolar macrophages and monocytes (Horwitz and Silverstein 1980). Several laboratories have begun a genetic analysis of L. pneumophila to identify the bacterial factors important in the interaction between legionellae and eukaroytic cells. However, these studies have been limited in scope as a result of the lack of a readily identifiable system of gene transfer for this organism. We (Mintz and Shuman 1988), as well as others (Chen et al. 1984; Dreyfus and Iglewski 1985), demonstrated that broad host range IncP plasmids, such as RK2, can be easily transferred by conjugation from Escherichia coli to L . pneumophila and between different strains of L. pneumophila. An important feature of IncP plasmids is the ability to mobilize the chromosomes of Gram-negative bacteria that lack endogenous systems of chromosome transfer (Barrett et al. 1982; Bittle and Konopka 1990; Towner 1978). Attempts to mobilize the L. pneumophila genome with plasmid RK2 have been unsuccessful (Mintz and Shuman 1988). IncP plasmids that contain insertion ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Lmprime au Canada
elements, such as bacteriophage Mu, exhibit an increased capacity as compared with native plasmids to promote the transfer of chromosomal DNA (Chatterjee et al. 1985; Lejeune et al. 1983; Van Gijsegem and Toussaint 1982). Several groups have taken advantage of this observation and have used Mu-mediated chromosome transfer to generate genetic linkage maps for a variety of Gram-negative bacteria (Forbes and Perombelon 1985; Schoonejans and Toussaint 1983). Although bacteriophage Mu and mini-Mu fusion phages are capable of replicative transposition in L. pneumophila (Mintz and Shuman 1987), the ability of IncP plasmids that contain Mu insertions to promote chromosome transfer in L. pneumophila has never been evaluated. Simon (1984) constructed the recombinant transposon Tn5-Mob by inserting the Mob site of plasmid RP4 into transposon Tn5. The insertion of Tn5-Mob into a replicon is sufficient to cause conjugal transfer of the DNA from one cell to another when the transfer functions of plasmid RP4 are supplied in trans. The Tn5-Mob system has been used to promote oriented, polar transfer of chromosomal genes in Rhizobium meliloti and E. coli (Simon 1984). The ability of Tn5 to transpose within the L. pneumophila genome (Keen and Hoffman 1985) suggested that Tn5-Mob may be useful for chromosome mobilization studies with L. pneumophila.
MINTZ AND ZOU
TABLE1. Bacterial strains, plasmids, and phages
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Strain, plasmid, or phage Strain L. pneumophila AM 170 CS 1 CS2 CS24 CS26 CS 123 CS 124 CS125 CS 126 CS138 CS220 CS291 CS293 CS295 CS297 CS302 CS303 JClO E. coli CMl00l DF5598 SMlO Plasmid RK2 pRK2 12 pRK24 pRK24.1 pRK24.4 pSUP5Oll Phage Mu cts62 Mu dl1681
Relevant properties"
Source or reference
CSl, r - m + Philadelphia- 1 Sm ' Bloomington-2 Rifr CSl (RK2) CSl (pRK212) CS1::Mu dI1681 No. 13 (pRK24.1) CS1::Mu dI1681 No. 22 (pRK24.1) CS1::Mu dI1681 No. 38 (pRK24.1) CS1::Mu dI1681 No. 46 (pRK24.1) Bloomington-2 thy Smr JClO thy AM170 (pRK24.4) AM 170::TnS-Mob (pRK24.4) AM 170::TnS-Mob (pRK24.4) AM170: :TnS-Mob (pRK24.4) Bloomington-2 trp Rifr Bloomington-2 gua Rifr Philadelphia-1 trp Rifr
Marra and Shuman 1989 Mintz and Shuman 1987 This study Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 This study This study This study This study This study This study This study This study Mintz et al. 1988
recA, bio thy::Mu cts62 recA, AtrpES leuB6 r - m recA leu supE contains integrated RP4-2-Tc::Mu Kmr
This study D. Figurski Simon et al. 1983
Apr Tcr Kmr RK2::Mu cts62 Apr Tcr Kmr RK2 neo: :[HindIII fragment] KmS pRK24: :Mu dI 1681 Kmr pRK24::Tn9 Cmr pBR325::TnS-Mob Kmr Apr Cmr
Meyer et al. 1977 Figurski et al. 1976 Figurski et al. 1976
+
Temperature-sensitive repressor Mini-Mu that carries lac operon of E. coli and Tn5 Kmr Mu cts62
This study This study Simon et al. 1983 M. Howe Castilho et al. 1984
'Smr, streptomycin resistant; Rifr, rifampicin resistant; r - , restriction minus; m', modification plus; A p r , ampicillin resistant; Tcr, tetracycline resistant; Kmr, kanamycin resistant; KmS, kanamycin sensitive; C m r , chloramphenicol resistant.
In the present report, we evaluated Mu-containing RK2 plasmids and Tn5-Mob chromosomal insertions for the ability to mobilize the L. pneumophila chromosome. The results from mating experiments showed that RK2::Mu plasmids and Tn5-Mob can promote the conjugal transfer of chromosomal genes in L. pneumophila. Materials and methods
Bacterial strains, plasmids, and Mu phages Bacterial strains, plasmids, and Mu phages used in this study are listed in Table 1. Media and growth conditions Legionella pneumophila was routinely grown on Aces-buffered charcoal yeast extract (ABCYE) agar or in albumin yeast extract (AYE) broth (Horwitz and Silverstein 1980). Escherichia coli strain SMlO was grown in Luria broth (LB). Unless otherwise specified, bacteria were grown at 37°C. Antibiotics were used at the following concentrations (pg/mL): streptomycin (Sm), 50; rifampicin (Rif), 25; kanamycin (Km), 25; ampicillin (Ap), 50; and chloramphenicol (Cm), 20. All antibiotics were obtained from Sigma Chemical Co., St. Louis, Mo.
Isolation of L. pneumophila auxotrophic mutants The formulation of CAA, a semidefined minimal medium that does not contain L-tryptophan, purines, pyrimidines, and a variety of vitamins, has permitted the isolation of auxotrophic mutants of L. pneumophila (Mintz et al. 1988). Tryptophan (Trp) and guanine (Gua) auxotrophs were isolated from strains Philadelphia-1 or Bloomington-2 by ethyl methanesulfonate mutagenesis followed by penicillin enrichment, according to the methods of Mintz et al. 1988. Trp- and Gua- mutants were identified by their failure to grow on CAA agar in the absence of added L-tryptophan or guanine. Trimethoprim selection (Mintz et al. 1988) was used to isolate thymidine (Thy) auxotrophs from Philadelphia-1 and Bloomington-2. Strain CS220 (a Thy- Trp- mutant of strain Philadelphia-1) was isolated by plating strain JClO (Trp-) on trimethoprim-containing CAA agar, as previously described (Mintz et al. 1988). Prior to use in mating experiments, the nutritional requirements of each of the auxotrophs were confirmed by growth on CAA medium supplemented with the appropriate amino acid or base. When required, amino acids and bases were supplemented at concentrations of 100 pg/mL. All of the auxotrophs used in this study had reversion frequencies of 1 1 0 -*. Construction of Mu-containing donors RK2 plasmids that contained insertions of Mu cts or mini-Mu
CAN. J. MICROBIOL. VOL. 38, 1992
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oriT
-:
(')A
Chromosome Mobilization
A
B
C
D
E
F Donor
F
Recipient
a
B
C
D
R' Formation
W
e
f
E
B
Recombinant
FIG. 1. Chromosome mobilization and R ' formation mediated by Mu-containing IncP plasmids. Integration of the Mu-containing episome into the bacterial chromosome occurs by Mu-mediated replicon fusion. The resulting cointegrate contains the integrated plasmids flanked by two copies of Mu in the same orientation (1) Cells that contain the cointegrate structure can transfer chromosomal markers during conjugation. The oriT of the integrated plasmid serves as the origin of chromosome transfer in these strains. Homologous recombination between donor and recipient chromosomal DNA gives rise to recombinants following conjugation. (2) As a result of the unstable nature of the cointegrate structure, Mu-mediated excision of the integrated plasmid from the bacterial chromosome frequently results in the formation of R ' plasmids. phage Mu d11681 were isolated as previously described (Mintz and Shuman 1987). These plasmids were transferred by conjugation from E. coli t o L. pneumophila wild-type strains CS1 (Philadelphia-1) and CS2 (Bloomington-2). Strains that contained Mu dI 1681 chromosomal insertions and plasmid pRK24.1 were previously isolated and characterized in this laboratory (Mintz and Shuman 1987). Legionellae that contained RK2: :Mu plasmids or Mu dI 1681 insertions were grown at 30°C since all of the Mu phages used in this study contained a thermolabile Mu repressor protein. Isolation of Tn5-Mob insertions in the L. pneumophila chromosome The mobilizable plasmid pSUP5011 (pBR325::TnS-Mob, Cmr, Apr Kmr) was mated from E. coli SMlO to L. pneumophila strain AM170 as previously described (Mintz and Shuman 1988). Strain AM170 is a derivative of strain Philadelphia-1 that is a more efficient recipient of plasmid DNA than the wild type in heterospecific matings (Marra and Shuman 1989). Transconjugants were removed from selection plates and grown in 20 mL AYE broth without antibiotics at 37°C for 24 h on a gyratory shaker. After incubation, 5 mL was removed from the culture and centrifuged at 10 000 x g for 10 min, and the resultant cell pellet was suspended in 20 mL of AYE broth without antibiotics and incubated at 37°C as described above. This procedure was repeated for three successive 24-h periods. Following the final incubation, the culture was plated for single colonies onto ABCYE agar that contained kanamycin. These plates were incubated at 37°C for 3 days and then replica plated onto ABCYE agar that contained either chloramphenicol or ampicillin. Three transconjugants that were resistant to Km but sensitive to Cm and Ap were chosen for further study. Chromosomal DNA was prepared from each of the transconjugants as previously described (Mintz and Shuman 1987), digested with EcoRI or BamHI (Promega, Madison Wis.), elec-
trophoresed in ethidium bromide agarose gels, and transferred to nitrocellulose paper (MSI, Westboro, Mass.). These blots were probed with either a 32~-labeled3.6-kb Hind111 fragment from Tn5 or 32~-labeled pBR322 DNA and then visualized by autoradiography with Kodak X-Omat Film (Kodak, Rochester, N.Y .). Mating procedures Cultures of donors or recipients were grown in AYE broth on a gyratory shaker to densities of ca. lo8 cfu/mL. All strains were grown at 37"C, except for Mu-containing strains, which were grown at 30°C. Cells were harvested by centrifugation at 6000 x g for 10 min at room temperature and suspended in 1 mL of M63 buffer (Silhavy et al. 1984) to densities of lo9 cfu/mL. Samples of donor (0.1 mL) and recipient (0.1 mL) were mixed together on the surface of an ABCYE agar plate. The plate was then dried at room temperature for 30 min and incubated for 18 h at either 30 or 37°C. Previous experiments performed in this laboratory indicated that 18 h of incubation was optimal for plasmid transfer during L. pneumophila matings (Mintz and Shuman 1987, 1988). After the incubation period, the mating mixture was scraped from the plate into 3 mL M63 buffer with a sterile microscope slide and centrifuged, and the resultant cell pellet was suspended in 1 mL M63 buffer. Serial dilutions were made in M63 buffer and samples were spread onto CAA agar that contained the appropriate antibiotic or nutritional supplement for enumeration. Mock crosses using donor or recipient alone were included in all experiments. In all cases, the donor strain was completely counterselected on our selective media. Recombination frequencies were calculated by dividing the number of exconjugants minus revertants (obtained from recipient alone crosses) by the total number of recipients present at the end of the matings. All mating experiments were repeated at least two times.
MINTZ AND ZOU
TABLE2. Mu-mediated chromosome mobilization of strains Bloomington-2 and Philadelphia- 1a Donor
Plasmid
Recipient
Selected marker
CSl CSl CS2 CS2 CS2
RK2 pRK212 RK2 pRK212 pRK24.1
JClO JClO CS 138 CS138 CS138
Trp Trp Thy+ Thy Thy
Recombination frequency
+
+
+
+
'Matings were performed at 30°C as described in the Material and methods. Trp+ exconjugants were selected on CAA agar that contained rifampicin. Thy exconjugants were selected on CAA agar plus streptomycin. b ~ u m b e rof Trp+ or Thy exconjugants divided by the total number of recipients. +
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+
Detection of R prime plasmids Exconjugants that contained plasmid antibiotic resistance markers were mated with E. coli thy or trp recipients to test for R prime plasmids by complementation. In these experiments, plasmid-bearing L. pneumophila donors and E. coli auxotrophic recipients were grown to early log phase in liquid medium, spotted onto ABCYE agar plates at a donor-recipient ratio of 1:1, and incubated at 30°C for 18 h. Escherichia coli Mu cts62 lysogens were used as recipients in these crosses to prevent zygotic induction during transfer of RK2::Mu plasmids. After incubation, the mating mixtures were removed from the plates, washed twice with M63 buffer, and suspended in 1 mL of M63 buffer. Serial dilutions were made in M63 salts buffer, and samples were spread onto M63-glucose agar that contained Km and Ap to select for transconjugants complemented to ~ r o t o t r o ~ Mating h ~ . mixtures were also plated on LB agar plus Km and AP to monitor plasmid transfer. Both sets of selection plates were incubated at 30°C for 48 h.
TABLE3. Effect of Mu dI1681 insertions on the transfer of chromosome markers in strain Philadelphia-la
Results RK2::Mu plasmids promote chromosome transfer Mu-mediated chromosome mobilization occurs by random integration of Mu-containing plasmids into the host genome via a process involving Mu-mediated replicon fusion (Fig. 1). The resulting Hfr-like cointegrates can be efficient donors of chromosomal markers using the oriT of the integrated plasmid as the origin of chromosome transfer. In some instances, Mu-mediated excision of the integrated plasmid can give rise to R ' plasmids that carry chromosomal genes that can be useful in complementation experiments. Since bacteriophage Mu is capable of replicative transposition in L. pneumophila, we reasoned that RK2::Mu plasmids could mobilize the L. pneumophila chromosome by a mechanism analogous to the one depicted in Fig. 1. To test this, mating experiments were performed between L. pneumophila donors that contained either RK2 or RK2::Mu plasmids and L. pneumophila auxotrophic recipients. Donors and recipients of either strain Philadelphia- 1 or strain Bloomington-2 were used in these experiments. In the crosses involving strain Bloomington-2, plasmidcontaining isolates of strain CS2 (Bloomington-2, Rifr) were used as donors and strain CS138 (Bloomington-2, Thy-) was used as a recipient. In the Philadelphia-1 matings, strain CS 1 (Philadelphia- 1, Smr) that contained RK2 or pRK212 was used as a donor, whereas strains JC10 (Philadelphia-1, Trp -) and CS220 (Philadelphia-1 , Trp Thy-) were used as recipients. Mu-containing plasmids pRK2 12 and pRK24.1 promoted the transfer of the Thy' phenotype in matings between wild-type Bloomington-2 donors and strain CS 138 (Thy - ).
The results from a representative experiment are given in Table 2. The frequency of transfer of the Thy' phenotype (ca. 6.0 x lop6)was 1000 times greater than the frequency of reversion to Thy ' by strain CS138 ( I 10 9 ) . There was no apparent difference in the chromosome mobilizing activity of either of the two Mu-containing plasmids. In contrast, Thy' exconjugants were not recovered from matings with Bloomington-2 donors that contained only plasmid RK2 (Table 2). This result confirmed our previous observation that plasmid RK2 cannot mobilize the L. pneumophila chromosome (Mintz and Shuman 1988). Plasmid pRK212 also promoted the transfer of the Thy' and Trp ' phenotypes in strain Philadelphia-1 . The results from representative experiments are presented in Tables 2 and 3. The frequency of Trp' transfer mediated by pRK212 was 2.5 x l o p 7 . This was 10-20 times greater than the frequency of reversion to Trp ' (ca. 3.0 x 10 -8) by recipient strains JClO and CS220. The recombination frequency for the thy locus in strain Philadelphia-1 was 4.0 x l o p 7 Thy' exconjugants per recipient (Table 3). Al.though the recombination frequency for the thy locus was appreciably lower in strain Philadelphia-1 as compared with strain Bloomington-2, it was 10 times greater than the frequency of reversion to Thy' by recipient strain CS220 ( I 3.4 x Similar to the results obtained from Bloomington-2 matings, Philadelphia-1 donors that contained plasmid RK2 did not transfer the thy or trp loci. Of interest, plasmid antibiotic-resistance markers were not detected in most of the Thy' or Trp' exconjugants recovered from the Philadelphia-1 or Bloomington-2 matings. Moreover, heterospecific matings between plasmid-
Recombination frequency Donor
Plasmid
Recipient
Thy
+
Trp
+
'Malings were lwrfromed at 30OC described in the Materials and methods. Thy + exconjugants were selected on CAA agar that contained L-tryptophan and Rif. Trp+ exconjugants were selected on CAA agar that contained thymidine and Rif. umber of Thy or Trp exconjugants divided by the total number of recipients. +
-
-
+
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CAN. J. MICROBIOL. VOL. 38,
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FIG. 2. Southern blot analysis of Tn5-Mob containing AM170 isolates. Genomic DNA was extracted from three different Tn5-Mob strains, digested with EcoRI or BamHI and hybridized with 32~-labeled 3.6-kb Hind111 fragment from Tn5. Tn5-Mob contains a single BamHI site and no EcoRI sites. (Lanes A-C) EcoRI-digested chromosomal DNA from Tn5-Mob strains 1-3; (lane D) EcoRI-digested pSUP5Oll DNA; (lanes E and F) same DNA as contained in lanes A-C digested with BamHI; (lane H) BamHI-digested chromosomal DNA from strain AM170. Numbers at left indicate the size of standard DNA fragments in kilobase pairs. bearing exconjugants and E. coli thy or trp recipients failed to reveal the presence of R ' plasmids (data not shown). This suggested that the phenotypes of these exconjugants resulted from chromosomal gene transfer followed by a homologous recombination event rather than from complementation by R ' plasmids.
Mu chromosomal insertions enhance chromosome transfer by RK2::Mu-containing donors One of the limitations of using Mu-containing plasmids to promote chromosome transfer is that the Hfr-like donors that are formed following Mu-mediated replicon fusion generally do not give polar, oriented transfer of chromosomal markers from a single fixed origin (Forbes and Perombelon 1985; Schoonejans and Toussaint 1983). Instead, chromosome transfer usually proceeds from multiple origins as a result of the random integration of Mu-containing plasmids into the chromosome of the donor population. In a previous study, we introduced several independent Mu dI 1681 insertions into the chromosome of strain Philadelphia-1 (Mintz and Shuman 1987). Strains that contained the Mu insertions also harbored pRK24.1 (pRK24::Mu d l 168l), the plasmid vector that was used to introduce Mu dI1681 into strain Philadelphia-1 . We reasoned that pRK24.1 found in each of these strains could, at a given frequency, integrate into the Philadelphia-1 chromosome at the site of the Mu dI 1681 insertion via homologous recombination. Legionellae that contained the integrated plasmid could then serve as donors in mating experiments and chromosome transfer presumably would proceed from a single fixed origin. Four isolates of strain CS1 that contained different Mu dI 1681 chromosomal insertions and pRK24.1 were evaluated for the ability to act as donors in matings experiments with strain CS220. Previous Southern hybridization experiments showed that each of the putative donors contained a single Mu dI 1681 insertion, and each of the insertions was located at a different site within the Philadelphia-1 chromosome (Mintz and Shuman 1987). Donors that did not contain Mu dI 1681 insertions but harbored plasrnids pRK2 12 (RK2: :Mu cts62) or RK2 served as positive and negative controls,
respectively. All of the strains that contained Mu dI1681 insertions and pRK24.1 transferred the Trp phenotype at a higher frequency than donors that contained pRK212 alone (Table 3). In contrast, only two of the donors, strains CS124 and CS 125, transferred the Thy phenotype during these matings. Of these two donors, only strain CS124 transferred the thy locus at a higher frequency than donors that harbored pRK212 alone. The results from these experiments showed that strains that contained Mu dI1681 chromosomal insertions plus pRK24.1 transferred the trp locus and, in one instance, the thy locus, at a higher frequency than those strains that harbored only RK2: :Mu plasmids . Additional mating experiments were performed between Philadelphia- 1 donors that contained pRK2 12 or Mu dI 1681 insertions (plus pRK24.1) and strain CS220 to determine .the cotransfer frequency of the Trp and Thy phenotypes. In these experiments, excoajugants were selected for the acquisition of a single marker, e.g., Trp +, and then screened for the inheritance of the second unselected marker, e.g., Thy + . In complementary matings, Thy + exconjugants were screened for inheritance of the Trp phenotype. In no instance did we observe cotransfer of the Thy + and Trp + markers during these matings (data not shown). +
+
+
+
+
Tn5-Mob insertions promote chromosome transfer in L. pneumophila Engleberg et al. (1988) showed that ColE 1 replicons can be maintained in L. pneumophila with antibiotic selection. However, in the absence of selection, these plasmids are spontaneously lost at a relatively high frequency. Therefore, we reasoned that a ColEl plasmid that carried Tn5-Mob would serve as a suitable suicide vector to introduce Tn5-Mob insertions into the L. pneumophila chromosome. T o test this possibility, we introduced the Tn.5-Mob containing ColEl plasmid pSUP5011 into L. pneumophila strain AM170. Using the selection strategy outlined in the Materials and methods section, we obtained three AM 170 isolates that contained Tn5-Mob insertions. Sou.thern hybridization experiments revealed that each of the isolates contained a single Tn5-Mob insertion, and that each of the insertions
MINTZ A N D ZOU
TABLE4. Tn5-Mob insertions promote chromosome transfer in L. pneumophilaa Donor
Recipient
Selected marker
Recombination frequency
'Matings were performed at 37OC as described in Materials and methods. Trp' exconjugants were selected on CAA agar plus Rif. b ~ r p ' or Gua' exconjugants divided by the total number o f recipients.
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was located at a different site in the AM170 chromosome (Fig. 2). The results from Southern hybridization experiments using pBR322 DNA as a probe showed that these strains did not contain plasmid DNA sequences (data not shown). This suggested that the Tn5-Mob insertions contained in .these strains resulted from a transposition event rather than from integration of pSUP5011 into the AM170 chromosome. To determine if Tn5-Mob containing donors can transfer chromosomal markers, we introduced helper plasmid pRK24.4 into each of the three Tn5-Mob strains. These strains, designated CS293, CS295, and CS297, were used as donors in matings with strain JC10. As previously mentioned, strain JClO is a Trp - mutant isolated from strain Philadelphia- 1 (Table 1). Strain CS291 (AM 170 that contained pRK24.4) served as a negative control in these experiments. Although we were able to detect conjugal transfer of pRK24.4, Trp + exconjugants were never recovered from these matings (data not shown). This result was surprising to us but not without explanation. The Tn5-Mob insertions were generated in strain AM170 (Marra and Shuman 1989), which has been reported to lack the DNA restriction activity of the LpnII restriction-modification system of L. pneumophila (Chen et al. 1984). Although strain AM170 is thought to maintain DNA modification activity, the possibility existed that the modification system of strain AM170 was defective. Therefore, the inability to recover Trp exconjugants from the matings using AM1 70 donors may have resulted from the restriction-proficient phenotype of recipient strain JC10. To test this idea, we performed mating experiments between Tn5-Mob containing donors and Bloomington-2 gua o r trp recipients. Strain Bloomington-2 is deficient in both DNA restriction and modification activity (Marra and Shuman 1989). We reasoned that the use of this strain background would be likely to overcome possible restriction problems encountered in the Philadelphia-1 matings. The results from these experiments showed that strain CS293 but not strains CS295 or CS297 transferred the Trp + and Gus+ phenotypes to Bloomington-2 auxotrophic recipients (Table 4). CS293 transferred both markers at a frequency approximately 10 times greater than strains that did not contain Tn5-Mob insertions. The recovery of prototrophic exconjugants from these matings suggested that our inability to recover Trp + recombinants from the original Philadelphia-1 matings probably resulted from the restriction-proficient phenotype of recipient strain JC10. Nonetheless, these results indicated that Tn5-Mob insertions can be used to mobilize the L. pneumophila chromosome. +
and Gua'
Discussion The ability to transfer chromosomal DNA between different strains of L. pneumophila would greatly aid studies concerned with the genetic organization and molecular biology of this organism. However, as previously mentioned, L. pneumophila has been reported to lack an endogenous system of chromosome transfer. In a previous study, Dreyfus and Iglewski 1985 reported that the IncP plasmid R68.45 promoted the transfer of a single chromosomal marker (Thy +) in L. pneumophila strain Knoxville-1. In the present study, we showed that RK2::Mu plasmids and Tn5-Mob can be used to promote the transfer of several chromosomal markers in L. pneumophila. Matings performed between wild-type donors that contained RK2::Mu plasmids and auxotrophic recipients showed that Mu-containing plasmids promoted the transfer of chromosomal markers in L. pneumophila strains Philadelphia-1 and Bloomington-2 (Tables 2 and 3). Chromosome transfer probably occurred by a mechanism involving Mu-mediated replicon fusion, as depicted in Fig. 1. Of interest, the recombination frequencies for selected markers, e.g., Thy + , were 10-fold lower in matings performed with donors and recipients from strain Philadelphia-1 as compared with strain Bloomington-2. At the present time, there is no clear explanation for this observation. It is possible that strain Philadelphia-1 is less efficient than strain Bloomington-2 at forming stable mating pairs during conjugation. Alternatively, bacteriophage Mu may transpose at a higher frequency in strain Bloomington-2 as compared with strain Philadelphia-1 . Consequently, RK2: :Mucontaining Bloomington-2 donors may have transferred .the Thy phenotype at a higher frequency than Philadelphia-1 donors as a result of an increase in the number of cells within the donor population that contained integrated copies of RK2::Mu. Nevertheless, our results clearly showed that RK2::Mu plasmids can mobilize the L. pneumophila chromosome. As shown in Fig. 1, the improper excision of Mu-containing plasmids from the bacterial chromosome during conjugation can result in the formation of R ' plasmids which carry chromosomal markers. Mu-containing IncP plasmids have been reported to mediate the formation of R ' s in a variety of Gram-negative bacteria (Chatterjee et al. 1985; Lejeune et al. 1983; Van Gijsegem and Toussaint 1982). Typically, R ' formation is detected by complementation using E. coli auxotrophic mutants as recipients in heterospecific matings. Matings between plasmid-bearing L. pneumophila Thy' or Trp' exconjugants and recA, restriction-deficient E. coli trp and thy recipients failed to +
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complement these mutants to prototrophy. These results suggested that R ' s were not responsible for the phenotypes of the Thy' and Trp exconjugants obtained from mating experiments. It is possible that our inability to complement the E. coli auxotrophs used in this study did not result from the lack of R ' formation in RK2::Mu-containing legionellae but, rather, from the poor expression of L. pneumophila trp or thy genes in E. coli. However, this is unlikely, since we have recently used complementation of E. coli trp mutants to identify and clone L. pneumophila trp genes (C. Mintz and C. Eddy, unpublished results). Several investigators have used transposon-facilitated recombination (Tfr) to mobilize the chromosomes of a variety of Gram-negative species (Deich and Green 1987; Ichige et al. 1989; Pischl and Farrand 1983). In this system, regions of DNA sequence homology are created between a conjugal plasmid and the bacterial chromosome of a donor strain by introducing identical transposable elements into each of the replicons. Tfr strains are thought to transfer chromosomal genes following integration of the plasmid via homologous recombination at the site of the chromosomal transposon insertion. To determine if this approach could be used t o promote chromosome transfer in L. pneumophila, we constructed Philadelphia-1 strains that contained Mu dI 1681 chromosomal insertions and plasmid pRK24.1 (pRK24::Mu dI1681). These strains transferred the Trp +,and, in one instance, the Thy+ phenotypes more efficiently than donors that contained only RK2::Mu plasmids (Table 3). As previously mentioned, chromosome transfer in donor populations that harbor Mu-containing IncP plasmids usually occurs from multiple origins. In contrast, the transfer of chromosomal markers in Tfr strains occurs from a single fixed origin. It is possible that this may have accounted for the increased efficiency with which Mu dI1681 Tfr donors transferred selected markers in our mating experiments. Differences in the ability of individual Mu d11681 Tfr strains to donate the Thy+ or Trp + markers may have resulted from differences in the location of the Mu d11681 chromosomal insertions relative to the thy or trp locus, i.e., strains that contained Mu dI168 1 insertions close to the selected marker, would have transferred the marker at a higher frequency than donors that contained the insertion at a more distant location. In support of this idea, we have previously shown by Southern hybridization that the Mu dI1681 insertion contained in each of the Tfr strains is found at a different site in the L. pneumophila chromosome (Mintz and Shuman 1987). Also, differences in .the direction of chromosome transfer may have affected the ability of individual Mu dI1681 Tfr donors to transfer selected markers. Our inability to detect cotransfer of the Thy and Trp phenotypes in matings between Mu dI 1681 Tfr donors and a Thy- Trp - recipient suggested that there was no detectable linkage between the thy and trp loci in strain Philadelphia- 1. Simon (1984) used the Tn5-Mob system to mobilize the chromosomes of E. coli and Rhizobium spp. In our study, a Tn5-Mob containing donor (CS293), in the presence of helper plasmid pRK24.4, transferred .the Trp and Gua phenotypes to auxotrophic recipients of strain Bloomington-2 (Table 2). Two other Tn5-Mob strains did not transfer these markers. The inability of these strains to transfer the Trp or Gua phenotypes could be explained by the location of Tn5-Mob relative to the trp or gua locus or by the direction
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of chromosome transfer in these strains. Currently, efforts are underway to introduce Tn5-Mob insertions into the Bloomington-2 chromosome to further characterize the chromosome mobilizing activity of Tn5-Mob in L. pneumophila. It is evident from our results that RK2::Mu plasmids and Tn5-Mob can be used to mobilize the L. pneumophila chromosome. Ultimately, we would like to use either or both of these systems t o construct a genetic map for L. pneumophila. However, to accomplish this, it will be necessary to identify additional genetic markers for L. pneumophila. As a result of the complex nutritional requirements of L. pneumophila and, consequently, the chemical composition of CAA medium, the types of auxotrophs that can be isolated from L. pneumophila are limited (Mintz et al. 1988). T o overcome this problem, we recently constructed donor and recipient strains that are resistant to a variety of antibiotics and amino acid analogues (C. Mintz, unpublished results). Therefore, as more genetic markers become available, it should be possible to use Tn5-Mob or Mu-mediated chromosome transfer to construct a rudimentary genetic map for L. pneumophila. Acknowledgements The authors thank Howard Shuman and David Figurski for helpful discussions, and Carleen Collins and Alan Kimura for critical review of the manuscript. The expert typing and graphic skills of Michelle Soto are gratefully acknowledged. Barrett, J.T., Rhodes, C.S., Ferber, D.M., et al. 1982. Construction of a genetic map for Caulobacter cresentus. J. Bacteriol. 149: 889-896. Beringer, J.E., Hoggan, S.A., and Johnston, A. W.B. 1978. Linkage mapping in Rhizobium leguminosarum by means of R plasmid-mediated recombination. J. Gen. Microbiol. 104: 20 1-207. Bittle, C., and Konopka, A. 1990. IncP-mediated transfer of loci involved with gas vesicle production in Ancylobacter aquaticus. J. Gen. Microbiol. 136: 1259-1263. Castilho, B., Olfson, P., and Casadaban, M.J. 1984. Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J. Bacteriol. 158: 488-495. Chatterjee, A.K., Ross, L.M., McEvoy, J.L., and Thurn, K.K. 1985. pULB 113, an RP4::mini-Mu plasmid, mediated chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia crysanthemi, and subspecies of Erwinia carotovora. Appl. Eviron. Microbiol. 50: 1-9. Chen, G.C., Lema, M., and Brown, A. 1984. Plasmid transfer into members of the family Legionellaceae. J. Infect. Dis. 150: 513-516. Chen, G.C., Brown, A., and Lema, M.W. 1986. Restriction endonuclease activities in the legionellae. Can. J. Microbiol. 32: 591-593. Deich, R.A., and Green, B.A. 1987. Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F ' factor. J. Bacteriol. 169: 1905-1910. Dreyfus, L.A. 1987. Virulence associated ingestion of Legionella pneumophila by HeLa cells. Microb. Pathog. 3: 45-52. Dreyfus, L.A., and Iglewski, B.H. 1985. Conjugation-mediated genetic exchange in Legionella pneumophila. J. Bacteriol. 161: 80-84. Engleberg, N.C., Cianciatto, N., Smith, J., and Eisenstein, B.I. 1988. Transfer and maintenance of small, mobilizable plasmids with ColEl replication origins in Legionella pneumophila. Plasmid, 20: 83-91.
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Fields, B.A., Barbaree, J.M., Shotts, E.B., et al. 1986. Comparison of guinea pig and protozoan models for determining the virulence of Legionella species. Infect. Immun. 53: 553-559. Figurski, D., Meyer, R., Miller, D.S., and Helinski, D.R. 1976. Generation of in vitro deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease. Gene, 1: 107-119. Forbes, K. J., and Perombelon, M.C.M. 1985. Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu. J. Bacteriol. 164: 1110-1 116. Hortwitz, M.A., and Silverstein, S.C. 1980. Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J . Clin. Invest. 66: 44.1-450. Ichige, A., Matsutani, S., Oishi, K., and Mizushima, S. 1989. Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain. J . Bacteriol. 171: 1825-1834. Keen, M.G., and Hoffman, P.S. 1985. Broad host range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome. J. Bacteriol. 162: 1331-1335. Lejeune, P., Mergeay, M., Van Gijsegem, F., et al. 1983. Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB 113(RP4: :Mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2. J. Bacteriol. 155: 1015-1026. Marra, A., and Shuman, H.A. 1989. Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J. Bacteriol. 171: 2238-2240. Meyer, R., Figurski, D., and Helinski, D.R. 1977. Physical and genetic studies with restriction endonucleases on the broad host range plasmid RK2. Mol. Gen. Genet. 152: 129-135. Mintz, C.S., and Shuman, H.A. 1987. Transposition of bacteriophage Mu in the legionnaires disease bacterium. Proc. Natl. Acad. Sci. U.S.A. 84: 4645-4649.
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Mintz, C.S., and Shuman, H.A. 1988. Genetics of Legionella pneumophila. Microbiol. Sci. 5: 292-295. Mintz, C.S., Chen, J., and Shuman, H.A. 1988. Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes. Infect. Immun. 56: 1449-1455. Oldham, L. J., and Rodgers, F.G. 1985. Adhesion, penetration and intracellular replication of Legionella pneumophila: an in vitro model of pathogenesis. J. Gen. Microbiol. 131: 697-706. Pischl, D.L., and Farrand, S.K. 1983. Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens. J . Bacteriol. 153: 1451-1460. Schoonejans, E., and Toussaint, A. 1983. Utilization of plasmid pULB113 (RP4::Mini-Mu) to construct a linkage map of Erwinia carotova subsp. chrysanthemi. J . Bacteriol. 154: 1489-1492. Silhavy, T. J., Berman, M.L., and Enquist, L. W. 1984. Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Simon, R. 1984. High frequency mobilization of gram negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol. Gen. Genet. 196: 413-420. Simon, R., Priefer, U., and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. BioTechniques, 1: 784-791. Towner, K. J. 1978. Chromosome mapping in Acinetobacter calcoaceticus J. Gen. Microbiol. 104: 175- 180. Van Gijsegem, F., and Toussaint, A. 1982. Chromosome transfer and R-prime formation by an RP4-mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Protease mirabilis. Plasmid, 7: 30-44.
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CLIFFORDS. MINTZ'AND CHANGH U A ZOU Department of Microbiology a n d Immunology, University of Miami, School of Medicine, P.O. Box 016960 (R-138), Miami, F L 33101, U.S.A. Received August 26, 1991 Revision received January 7, 1992 Accepted January 20, 1992 MINTZ, C. S., and Z o u , C . H. 1992. Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob. Can. J. Microbiol. 38: 664-671. RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionellapneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded per recipient for the markers tested. The presence of a Mu to recombinants at fequencies ranging from insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromoscme transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mucontaining donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila. Key words: Legionella pneumophila, chromosome transfer, Tn5-Mob, RK2::Mu. MINTZ, C. S., et Z o u , C. H. 1992. Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob. Can. J. Microbiol. 38 : 664-671. Des plasmides RK2::Mu et le transposon Tn5-Mob ont etk utilisks pour mobiliser le chromosome de Legionellapneumophila. Des conjugaisons en plaque entre les L. pneumophila donneurs contenant les plasmides RK2::Mu et les recepar receveur pour les marqueurs a veurs auxotrophes ont produit des recombinants a des frkquences de analyses. La presence d'une insertion Mu dans le chromosome des donneurs portant les plasmides RK2::Mu a augmentk la frequence du transfert chromosomique de certains marqueurs sklectionnes en comparaison avec les souches qui contenaient RK2::Mu seulement. Les experiences de cotransfert avec les donneurs contenant Mu et un auxotrophe pour la thymidine et le tryptophane n'ont pas permis de reveler un lien entre les locus thy et trp chez L. pneumophila. Une souche qui contenait une insertion chromosomique Tn5-Mob et le plasmide auxiliaire pRK24.4 a transfkrk les marqueurs chromosomiques a des frkquences de 10 - 7 par receveur. Ces rksultats sugghent que les plasmides RK2::Mu et le transposon Tn5-Mob peuvent 2tre utiles pour les experiences de cartographie genktique de L. pneumophila. Mots elks : Legionella pneumophila, transfert chromosomique, Tn5-Mob, RK2::Mu. [Traduit par la redaction]
Introduction Legionella pneumophila is a facultative intracellular pathogen capable of entering and growing in a variety of eukaryotic cells, including free-living amoebae (Fields et al. 1986), cultured animal cells (Dreyfus 1987; Oldham and Rodgers 1985), and alveolar macrophages and monocytes (Horwitz and Silverstein 1980). Several laboratories have begun a genetic analysis of L. pneumophila to identify the bacterial factors important in the interaction between legionellae and eukaroytic cells. However, these studies have been limited in scope as a result of the lack of a readily identifiable system of gene transfer for this organism. We (Mintz and Shuman 1988), as well as others (Chen et al. 1984; Dreyfus and Iglewski 1985), demonstrated that broad host range IncP plasmids, such as RK2, can be easily transferred by conjugation from Escherichia coli to L . pneumophila and between different strains of L. pneumophila. An important feature of IncP plasmids is the ability to mobilize the chromosomes of Gram-negative bacteria that lack endogenous systems of chromosome transfer (Barrett et al. 1982; Bittle and Konopka 1990; Towner 1978). Attempts to mobilize the L. pneumophila genome with plasmid RK2 have been unsuccessful (Mintz and Shuman 1988). IncP plasmids that contain insertion ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Lmprime au Canada
elements, such as bacteriophage Mu, exhibit an increased capacity as compared with native plasmids to promote the transfer of chromosomal DNA (Chatterjee et al. 1985; Lejeune et al. 1983; Van Gijsegem and Toussaint 1982). Several groups have taken advantage of this observation and have used Mu-mediated chromosome transfer to generate genetic linkage maps for a variety of Gram-negative bacteria (Forbes and Perombelon 1985; Schoonejans and Toussaint 1983). Although bacteriophage Mu and mini-Mu fusion phages are capable of replicative transposition in L. pneumophila (Mintz and Shuman 1987), the ability of IncP plasmids that contain Mu insertions to promote chromosome transfer in L. pneumophila has never been evaluated. Simon (1984) constructed the recombinant transposon Tn5-Mob by inserting the Mob site of plasmid RP4 into transposon Tn5. The insertion of Tn5-Mob into a replicon is sufficient to cause conjugal transfer of the DNA from one cell to another when the transfer functions of plasmid RP4 are supplied in trans. The Tn5-Mob system has been used to promote oriented, polar transfer of chromosomal genes in Rhizobium meliloti and E. coli (Simon 1984). The ability of Tn5 to transpose within the L. pneumophila genome (Keen and Hoffman 1985) suggested that Tn5-Mob may be useful for chromosome mobilization studies with L. pneumophila.
MINTZ AND ZOU
TABLE1. Bacterial strains, plasmids, and phages
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Strain, plasmid, or phage Strain L. pneumophila AM 170 CS 1 CS2 CS24 CS26 CS 123 CS 124 CS125 CS 126 CS138 CS220 CS291 CS293 CS295 CS297 CS302 CS303 JClO E. coli CMl00l DF5598 SMlO Plasmid RK2 pRK2 12 pRK24 pRK24.1 pRK24.4 pSUP5Oll Phage Mu cts62 Mu dl1681
Relevant properties"
Source or reference
CSl, r - m + Philadelphia- 1 Sm ' Bloomington-2 Rifr CSl (RK2) CSl (pRK212) CS1::Mu dI1681 No. 13 (pRK24.1) CS1::Mu dI1681 No. 22 (pRK24.1) CS1::Mu dI1681 No. 38 (pRK24.1) CS1::Mu dI1681 No. 46 (pRK24.1) Bloomington-2 thy Smr JClO thy AM170 (pRK24.4) AM 170::TnS-Mob (pRK24.4) AM 170::TnS-Mob (pRK24.4) AM170: :TnS-Mob (pRK24.4) Bloomington-2 trp Rifr Bloomington-2 gua Rifr Philadelphia-1 trp Rifr
Marra and Shuman 1989 Mintz and Shuman 1987 This study Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 Mintz and Shuman 1987 This study This study This study This study This study This study This study This study Mintz et al. 1988
recA, bio thy::Mu cts62 recA, AtrpES leuB6 r - m recA leu supE contains integrated RP4-2-Tc::Mu Kmr
This study D. Figurski Simon et al. 1983
Apr Tcr Kmr RK2::Mu cts62 Apr Tcr Kmr RK2 neo: :[HindIII fragment] KmS pRK24: :Mu dI 1681 Kmr pRK24::Tn9 Cmr pBR325::TnS-Mob Kmr Apr Cmr
Meyer et al. 1977 Figurski et al. 1976 Figurski et al. 1976
+
Temperature-sensitive repressor Mini-Mu that carries lac operon of E. coli and Tn5 Kmr Mu cts62
This study This study Simon et al. 1983 M. Howe Castilho et al. 1984
'Smr, streptomycin resistant; Rifr, rifampicin resistant; r - , restriction minus; m', modification plus; A p r , ampicillin resistant; Tcr, tetracycline resistant; Kmr, kanamycin resistant; KmS, kanamycin sensitive; C m r , chloramphenicol resistant.
In the present report, we evaluated Mu-containing RK2 plasmids and Tn5-Mob chromosomal insertions for the ability to mobilize the L. pneumophila chromosome. The results from mating experiments showed that RK2::Mu plasmids and Tn5-Mob can promote the conjugal transfer of chromosomal genes in L. pneumophila. Materials and methods
Bacterial strains, plasmids, and Mu phages Bacterial strains, plasmids, and Mu phages used in this study are listed in Table 1. Media and growth conditions Legionella pneumophila was routinely grown on Aces-buffered charcoal yeast extract (ABCYE) agar or in albumin yeast extract (AYE) broth (Horwitz and Silverstein 1980). Escherichia coli strain SMlO was grown in Luria broth (LB). Unless otherwise specified, bacteria were grown at 37°C. Antibiotics were used at the following concentrations (pg/mL): streptomycin (Sm), 50; rifampicin (Rif), 25; kanamycin (Km), 25; ampicillin (Ap), 50; and chloramphenicol (Cm), 20. All antibiotics were obtained from Sigma Chemical Co., St. Louis, Mo.
Isolation of L. pneumophila auxotrophic mutants The formulation of CAA, a semidefined minimal medium that does not contain L-tryptophan, purines, pyrimidines, and a variety of vitamins, has permitted the isolation of auxotrophic mutants of L. pneumophila (Mintz et al. 1988). Tryptophan (Trp) and guanine (Gua) auxotrophs were isolated from strains Philadelphia-1 or Bloomington-2 by ethyl methanesulfonate mutagenesis followed by penicillin enrichment, according to the methods of Mintz et al. 1988. Trp- and Gua- mutants were identified by their failure to grow on CAA agar in the absence of added L-tryptophan or guanine. Trimethoprim selection (Mintz et al. 1988) was used to isolate thymidine (Thy) auxotrophs from Philadelphia-1 and Bloomington-2. Strain CS220 (a Thy- Trp- mutant of strain Philadelphia-1) was isolated by plating strain JClO (Trp-) on trimethoprim-containing CAA agar, as previously described (Mintz et al. 1988). Prior to use in mating experiments, the nutritional requirements of each of the auxotrophs were confirmed by growth on CAA medium supplemented with the appropriate amino acid or base. When required, amino acids and bases were supplemented at concentrations of 100 pg/mL. All of the auxotrophs used in this study had reversion frequencies of 1 1 0 -*. Construction of Mu-containing donors RK2 plasmids that contained insertions of Mu cts or mini-Mu
CAN. J. MICROBIOL. VOL. 38, 1992
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oriT
-:
(')A
Chromosome Mobilization
A
B
C
D
E
F Donor
F
Recipient
a
B
C
D
R' Formation
W
e
f
E
B
Recombinant
FIG. 1. Chromosome mobilization and R ' formation mediated by Mu-containing IncP plasmids. Integration of the Mu-containing episome into the bacterial chromosome occurs by Mu-mediated replicon fusion. The resulting cointegrate contains the integrated plasmids flanked by two copies of Mu in the same orientation (1) Cells that contain the cointegrate structure can transfer chromosomal markers during conjugation. The oriT of the integrated plasmid serves as the origin of chromosome transfer in these strains. Homologous recombination between donor and recipient chromosomal DNA gives rise to recombinants following conjugation. (2) As a result of the unstable nature of the cointegrate structure, Mu-mediated excision of the integrated plasmid from the bacterial chromosome frequently results in the formation of R ' plasmids. phage Mu d11681 were isolated as previously described (Mintz and Shuman 1987). These plasmids were transferred by conjugation from E. coli t o L. pneumophila wild-type strains CS1 (Philadelphia-1) and CS2 (Bloomington-2). Strains that contained Mu dI 1681 chromosomal insertions and plasmid pRK24.1 were previously isolated and characterized in this laboratory (Mintz and Shuman 1987). Legionellae that contained RK2: :Mu plasmids or Mu dI 1681 insertions were grown at 30°C since all of the Mu phages used in this study contained a thermolabile Mu repressor protein. Isolation of Tn5-Mob insertions in the L. pneumophila chromosome The mobilizable plasmid pSUP5011 (pBR325::TnS-Mob, Cmr, Apr Kmr) was mated from E. coli SMlO to L. pneumophila strain AM170 as previously described (Mintz and Shuman 1988). Strain AM170 is a derivative of strain Philadelphia-1 that is a more efficient recipient of plasmid DNA than the wild type in heterospecific matings (Marra and Shuman 1989). Transconjugants were removed from selection plates and grown in 20 mL AYE broth without antibiotics at 37°C for 24 h on a gyratory shaker. After incubation, 5 mL was removed from the culture and centrifuged at 10 000 x g for 10 min, and the resultant cell pellet was suspended in 20 mL of AYE broth without antibiotics and incubated at 37°C as described above. This procedure was repeated for three successive 24-h periods. Following the final incubation, the culture was plated for single colonies onto ABCYE agar that contained kanamycin. These plates were incubated at 37°C for 3 days and then replica plated onto ABCYE agar that contained either chloramphenicol or ampicillin. Three transconjugants that were resistant to Km but sensitive to Cm and Ap were chosen for further study. Chromosomal DNA was prepared from each of the transconjugants as previously described (Mintz and Shuman 1987), digested with EcoRI or BamHI (Promega, Madison Wis.), elec-
trophoresed in ethidium bromide agarose gels, and transferred to nitrocellulose paper (MSI, Westboro, Mass.). These blots were probed with either a 32~-labeled3.6-kb Hind111 fragment from Tn5 or 32~-labeled pBR322 DNA and then visualized by autoradiography with Kodak X-Omat Film (Kodak, Rochester, N.Y .). Mating procedures Cultures of donors or recipients were grown in AYE broth on a gyratory shaker to densities of ca. lo8 cfu/mL. All strains were grown at 37"C, except for Mu-containing strains, which were grown at 30°C. Cells were harvested by centrifugation at 6000 x g for 10 min at room temperature and suspended in 1 mL of M63 buffer (Silhavy et al. 1984) to densities of lo9 cfu/mL. Samples of donor (0.1 mL) and recipient (0.1 mL) were mixed together on the surface of an ABCYE agar plate. The plate was then dried at room temperature for 30 min and incubated for 18 h at either 30 or 37°C. Previous experiments performed in this laboratory indicated that 18 h of incubation was optimal for plasmid transfer during L. pneumophila matings (Mintz and Shuman 1987, 1988). After the incubation period, the mating mixture was scraped from the plate into 3 mL M63 buffer with a sterile microscope slide and centrifuged, and the resultant cell pellet was suspended in 1 mL M63 buffer. Serial dilutions were made in M63 buffer and samples were spread onto CAA agar that contained the appropriate antibiotic or nutritional supplement for enumeration. Mock crosses using donor or recipient alone were included in all experiments. In all cases, the donor strain was completely counterselected on our selective media. Recombination frequencies were calculated by dividing the number of exconjugants minus revertants (obtained from recipient alone crosses) by the total number of recipients present at the end of the matings. All mating experiments were repeated at least two times.
MINTZ AND ZOU
TABLE2. Mu-mediated chromosome mobilization of strains Bloomington-2 and Philadelphia- 1a Donor
Plasmid
Recipient
Selected marker
CSl CSl CS2 CS2 CS2
RK2 pRK212 RK2 pRK212 pRK24.1
JClO JClO CS 138 CS138 CS138
Trp Trp Thy+ Thy Thy
Recombination frequency
+
+
+
+
'Matings were performed at 30°C as described in the Material and methods. Trp+ exconjugants were selected on CAA agar that contained rifampicin. Thy exconjugants were selected on CAA agar plus streptomycin. b ~ u m b e rof Trp+ or Thy exconjugants divided by the total number of recipients. +
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+
Detection of R prime plasmids Exconjugants that contained plasmid antibiotic resistance markers were mated with E. coli thy or trp recipients to test for R prime plasmids by complementation. In these experiments, plasmid-bearing L. pneumophila donors and E. coli auxotrophic recipients were grown to early log phase in liquid medium, spotted onto ABCYE agar plates at a donor-recipient ratio of 1:1, and incubated at 30°C for 18 h. Escherichia coli Mu cts62 lysogens were used as recipients in these crosses to prevent zygotic induction during transfer of RK2::Mu plasmids. After incubation, the mating mixtures were removed from the plates, washed twice with M63 buffer, and suspended in 1 mL of M63 buffer. Serial dilutions were made in M63 salts buffer, and samples were spread onto M63-glucose agar that contained Km and Ap to select for transconjugants complemented to ~ r o t o t r o ~ Mating h ~ . mixtures were also plated on LB agar plus Km and AP to monitor plasmid transfer. Both sets of selection plates were incubated at 30°C for 48 h.
TABLE3. Effect of Mu dI1681 insertions on the transfer of chromosome markers in strain Philadelphia-la
Results RK2::Mu plasmids promote chromosome transfer Mu-mediated chromosome mobilization occurs by random integration of Mu-containing plasmids into the host genome via a process involving Mu-mediated replicon fusion (Fig. 1). The resulting Hfr-like cointegrates can be efficient donors of chromosomal markers using the oriT of the integrated plasmid as the origin of chromosome transfer. In some instances, Mu-mediated excision of the integrated plasmid can give rise to R ' plasmids that carry chromosomal genes that can be useful in complementation experiments. Since bacteriophage Mu is capable of replicative transposition in L. pneumophila, we reasoned that RK2::Mu plasmids could mobilize the L. pneumophila chromosome by a mechanism analogous to the one depicted in Fig. 1. To test this, mating experiments were performed between L. pneumophila donors that contained either RK2 or RK2::Mu plasmids and L. pneumophila auxotrophic recipients. Donors and recipients of either strain Philadelphia- 1 or strain Bloomington-2 were used in these experiments. In the crosses involving strain Bloomington-2, plasmidcontaining isolates of strain CS2 (Bloomington-2, Rifr) were used as donors and strain CS138 (Bloomington-2, Thy-) was used as a recipient. In the Philadelphia-1 matings, strain CS 1 (Philadelphia- 1, Smr) that contained RK2 or pRK212 was used as a donor, whereas strains JC10 (Philadelphia-1, Trp -) and CS220 (Philadelphia-1 , Trp Thy-) were used as recipients. Mu-containing plasmids pRK2 12 and pRK24.1 promoted the transfer of the Thy' phenotype in matings between wild-type Bloomington-2 donors and strain CS 138 (Thy - ).
The results from a representative experiment are given in Table 2. The frequency of transfer of the Thy' phenotype (ca. 6.0 x lop6)was 1000 times greater than the frequency of reversion to Thy ' by strain CS138 ( I 10 9 ) . There was no apparent difference in the chromosome mobilizing activity of either of the two Mu-containing plasmids. In contrast, Thy' exconjugants were not recovered from matings with Bloomington-2 donors that contained only plasmid RK2 (Table 2). This result confirmed our previous observation that plasmid RK2 cannot mobilize the L. pneumophila chromosome (Mintz and Shuman 1988). Plasmid pRK212 also promoted the transfer of the Thy' and Trp ' phenotypes in strain Philadelphia-1 . The results from representative experiments are presented in Tables 2 and 3. The frequency of Trp' transfer mediated by pRK212 was 2.5 x l o p 7 . This was 10-20 times greater than the frequency of reversion to Trp ' (ca. 3.0 x 10 -8) by recipient strains JClO and CS220. The recombination frequency for the thy locus in strain Philadelphia-1 was 4.0 x l o p 7 Thy' exconjugants per recipient (Table 3). Al.though the recombination frequency for the thy locus was appreciably lower in strain Philadelphia-1 as compared with strain Bloomington-2, it was 10 times greater than the frequency of reversion to Thy' by recipient strain CS220 ( I 3.4 x Similar to the results obtained from Bloomington-2 matings, Philadelphia-1 donors that contained plasmid RK2 did not transfer the thy or trp loci. Of interest, plasmid antibiotic-resistance markers were not detected in most of the Thy' or Trp' exconjugants recovered from the Philadelphia-1 or Bloomington-2 matings. Moreover, heterospecific matings between plasmid-
Recombination frequency Donor
Plasmid
Recipient
Thy
+
Trp
+
'Malings were lwrfromed at 30OC described in the Materials and methods. Thy + exconjugants were selected on CAA agar that contained L-tryptophan and Rif. Trp+ exconjugants were selected on CAA agar that contained thymidine and Rif. umber of Thy or Trp exconjugants divided by the total number of recipients. +
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CAN. J. MICROBIOL. VOL. 38,
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FIG. 2. Southern blot analysis of Tn5-Mob containing AM170 isolates. Genomic DNA was extracted from three different Tn5-Mob strains, digested with EcoRI or BamHI and hybridized with 32~-labeled 3.6-kb Hind111 fragment from Tn5. Tn5-Mob contains a single BamHI site and no EcoRI sites. (Lanes A-C) EcoRI-digested chromosomal DNA from Tn5-Mob strains 1-3; (lane D) EcoRI-digested pSUP5Oll DNA; (lanes E and F) same DNA as contained in lanes A-C digested with BamHI; (lane H) BamHI-digested chromosomal DNA from strain AM170. Numbers at left indicate the size of standard DNA fragments in kilobase pairs. bearing exconjugants and E. coli thy or trp recipients failed to reveal the presence of R ' plasmids (data not shown). This suggested that the phenotypes of these exconjugants resulted from chromosomal gene transfer followed by a homologous recombination event rather than from complementation by R ' plasmids.
Mu chromosomal insertions enhance chromosome transfer by RK2::Mu-containing donors One of the limitations of using Mu-containing plasmids to promote chromosome transfer is that the Hfr-like donors that are formed following Mu-mediated replicon fusion generally do not give polar, oriented transfer of chromosomal markers from a single fixed origin (Forbes and Perombelon 1985; Schoonejans and Toussaint 1983). Instead, chromosome transfer usually proceeds from multiple origins as a result of the random integration of Mu-containing plasmids into the chromosome of the donor population. In a previous study, we introduced several independent Mu dI 1681 insertions into the chromosome of strain Philadelphia-1 (Mintz and Shuman 1987). Strains that contained the Mu insertions also harbored pRK24.1 (pRK24::Mu d l 168l), the plasmid vector that was used to introduce Mu dI1681 into strain Philadelphia-1 . We reasoned that pRK24.1 found in each of these strains could, at a given frequency, integrate into the Philadelphia-1 chromosome at the site of the Mu dI 1681 insertion via homologous recombination. Legionellae that contained the integrated plasmid could then serve as donors in mating experiments and chromosome transfer presumably would proceed from a single fixed origin. Four isolates of strain CS1 that contained different Mu dI 1681 chromosomal insertions and pRK24.1 were evaluated for the ability to act as donors in matings experiments with strain CS220. Previous Southern hybridization experiments showed that each of the putative donors contained a single Mu dI 1681 insertion, and each of the insertions was located at a different site within the Philadelphia-1 chromosome (Mintz and Shuman 1987). Donors that did not contain Mu dI 1681 insertions but harbored plasrnids pRK2 12 (RK2: :Mu cts62) or RK2 served as positive and negative controls,
respectively. All of the strains that contained Mu dI1681 insertions and pRK24.1 transferred the Trp phenotype at a higher frequency than donors that contained pRK212 alone (Table 3). In contrast, only two of the donors, strains CS124 and CS 125, transferred the Thy phenotype during these matings. Of these two donors, only strain CS124 transferred the thy locus at a higher frequency than donors that harbored pRK212 alone. The results from these experiments showed that strains that contained Mu dI1681 chromosomal insertions plus pRK24.1 transferred the trp locus and, in one instance, the thy locus, at a higher frequency than those strains that harbored only RK2: :Mu plasmids . Additional mating experiments were performed between Philadelphia- 1 donors that contained pRK2 12 or Mu dI 1681 insertions (plus pRK24.1) and strain CS220 to determine .the cotransfer frequency of the Trp and Thy phenotypes. In these experiments, excoajugants were selected for the acquisition of a single marker, e.g., Trp +, and then screened for the inheritance of the second unselected marker, e.g., Thy + . In complementary matings, Thy + exconjugants were screened for inheritance of the Trp phenotype. In no instance did we observe cotransfer of the Thy + and Trp + markers during these matings (data not shown). +
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Tn5-Mob insertions promote chromosome transfer in L. pneumophila Engleberg et al. (1988) showed that ColE 1 replicons can be maintained in L. pneumophila with antibiotic selection. However, in the absence of selection, these plasmids are spontaneously lost at a relatively high frequency. Therefore, we reasoned that a ColEl plasmid that carried Tn5-Mob would serve as a suitable suicide vector to introduce Tn5-Mob insertions into the L. pneumophila chromosome. T o test this possibility, we introduced the Tn.5-Mob containing ColEl plasmid pSUP5011 into L. pneumophila strain AM170. Using the selection strategy outlined in the Materials and methods section, we obtained three AM 170 isolates that contained Tn5-Mob insertions. Sou.thern hybridization experiments revealed that each of the isolates contained a single Tn5-Mob insertion, and that each of the insertions
MINTZ A N D ZOU
TABLE4. Tn5-Mob insertions promote chromosome transfer in L. pneumophilaa Donor
Recipient
Selected marker
Recombination frequency
'Matings were performed at 37OC as described in Materials and methods. Trp' exconjugants were selected on CAA agar plus Rif. b ~ r p ' or Gua' exconjugants divided by the total number o f recipients.
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was located at a different site in the AM170 chromosome (Fig. 2). The results from Southern hybridization experiments using pBR322 DNA as a probe showed that these strains did not contain plasmid DNA sequences (data not shown). This suggested that the Tn5-Mob insertions contained in .these strains resulted from a transposition event rather than from integration of pSUP5011 into the AM170 chromosome. To determine if Tn5-Mob containing donors can transfer chromosomal markers, we introduced helper plasmid pRK24.4 into each of the three Tn5-Mob strains. These strains, designated CS293, CS295, and CS297, were used as donors in matings with strain JC10. As previously mentioned, strain JClO is a Trp - mutant isolated from strain Philadelphia- 1 (Table 1). Strain CS291 (AM 170 that contained pRK24.4) served as a negative control in these experiments. Although we were able to detect conjugal transfer of pRK24.4, Trp + exconjugants were never recovered from these matings (data not shown). This result was surprising to us but not without explanation. The Tn5-Mob insertions were generated in strain AM170 (Marra and Shuman 1989), which has been reported to lack the DNA restriction activity of the LpnII restriction-modification system of L. pneumophila (Chen et al. 1984). Although strain AM170 is thought to maintain DNA modification activity, the possibility existed that the modification system of strain AM170 was defective. Therefore, the inability to recover Trp exconjugants from the matings using AM1 70 donors may have resulted from the restriction-proficient phenotype of recipient strain JC10. To test this idea, we performed mating experiments between Tn5-Mob containing donors and Bloomington-2 gua o r trp recipients. Strain Bloomington-2 is deficient in both DNA restriction and modification activity (Marra and Shuman 1989). We reasoned that the use of this strain background would be likely to overcome possible restriction problems encountered in the Philadelphia-1 matings. The results from these experiments showed that strain CS293 but not strains CS295 or CS297 transferred the Trp + and Gus+ phenotypes to Bloomington-2 auxotrophic recipients (Table 4). CS293 transferred both markers at a frequency approximately 10 times greater than strains that did not contain Tn5-Mob insertions. The recovery of prototrophic exconjugants from these matings suggested that our inability to recover Trp + recombinants from the original Philadelphia-1 matings probably resulted from the restriction-proficient phenotype of recipient strain JC10. Nonetheless, these results indicated that Tn5-Mob insertions can be used to mobilize the L. pneumophila chromosome. +
and Gua'
Discussion The ability to transfer chromosomal DNA between different strains of L. pneumophila would greatly aid studies concerned with the genetic organization and molecular biology of this organism. However, as previously mentioned, L. pneumophila has been reported to lack an endogenous system of chromosome transfer. In a previous study, Dreyfus and Iglewski 1985 reported that the IncP plasmid R68.45 promoted the transfer of a single chromosomal marker (Thy +) in L. pneumophila strain Knoxville-1. In the present study, we showed that RK2::Mu plasmids and Tn5-Mob can be used to promote the transfer of several chromosomal markers in L. pneumophila. Matings performed between wild-type donors that contained RK2::Mu plasmids and auxotrophic recipients showed that Mu-containing plasmids promoted the transfer of chromosomal markers in L. pneumophila strains Philadelphia-1 and Bloomington-2 (Tables 2 and 3). Chromosome transfer probably occurred by a mechanism involving Mu-mediated replicon fusion, as depicted in Fig. 1. Of interest, the recombination frequencies for selected markers, e.g., Thy + , were 10-fold lower in matings performed with donors and recipients from strain Philadelphia-1 as compared with strain Bloomington-2. At the present time, there is no clear explanation for this observation. It is possible that strain Philadelphia-1 is less efficient than strain Bloomington-2 at forming stable mating pairs during conjugation. Alternatively, bacteriophage Mu may transpose at a higher frequency in strain Bloomington-2 as compared with strain Philadelphia-1 . Consequently, RK2: :Mucontaining Bloomington-2 donors may have transferred .the Thy phenotype at a higher frequency than Philadelphia-1 donors as a result of an increase in the number of cells within the donor population that contained integrated copies of RK2::Mu. Nevertheless, our results clearly showed that RK2::Mu plasmids can mobilize the L. pneumophila chromosome. As shown in Fig. 1, the improper excision of Mu-containing plasmids from the bacterial chromosome during conjugation can result in the formation of R ' plasmids which carry chromosomal markers. Mu-containing IncP plasmids have been reported to mediate the formation of R ' s in a variety of Gram-negative bacteria (Chatterjee et al. 1985; Lejeune et al. 1983; Van Gijsegem and Toussaint 1982). Typically, R ' formation is detected by complementation using E. coli auxotrophic mutants as recipients in heterospecific matings. Matings between plasmid-bearing L. pneumophila Thy' or Trp' exconjugants and recA, restriction-deficient E. coli trp and thy recipients failed to +
CAN. J. MICROBIOL.
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complement these mutants to prototrophy. These results suggested that R ' s were not responsible for the phenotypes of the Thy' and Trp exconjugants obtained from mating experiments. It is possible that our inability to complement the E. coli auxotrophs used in this study did not result from the lack of R ' formation in RK2::Mu-containing legionellae but, rather, from the poor expression of L. pneumophila trp or thy genes in E. coli. However, this is unlikely, since we have recently used complementation of E. coli trp mutants to identify and clone L. pneumophila trp genes (C. Mintz and C. Eddy, unpublished results). Several investigators have used transposon-facilitated recombination (Tfr) to mobilize the chromosomes of a variety of Gram-negative species (Deich and Green 1987; Ichige et al. 1989; Pischl and Farrand 1983). In this system, regions of DNA sequence homology are created between a conjugal plasmid and the bacterial chromosome of a donor strain by introducing identical transposable elements into each of the replicons. Tfr strains are thought to transfer chromosomal genes following integration of the plasmid via homologous recombination at the site of the chromosomal transposon insertion. To determine if this approach could be used t o promote chromosome transfer in L. pneumophila, we constructed Philadelphia-1 strains that contained Mu dI 1681 chromosomal insertions and plasmid pRK24.1 (pRK24::Mu dI1681). These strains transferred the Trp +,and, in one instance, the Thy+ phenotypes more efficiently than donors that contained only RK2::Mu plasmids (Table 3). As previously mentioned, chromosome transfer in donor populations that harbor Mu-containing IncP plasmids usually occurs from multiple origins. In contrast, the transfer of chromosomal markers in Tfr strains occurs from a single fixed origin. It is possible that this may have accounted for the increased efficiency with which Mu dI1681 Tfr donors transferred selected markers in our mating experiments. Differences in the ability of individual Mu d11681 Tfr strains to donate the Thy+ or Trp + markers may have resulted from differences in the location of the Mu d11681 chromosomal insertions relative to the thy or trp locus, i.e., strains that contained Mu dI168 1 insertions close to the selected marker, would have transferred the marker at a higher frequency than donors that contained the insertion at a more distant location. In support of this idea, we have previously shown by Southern hybridization that the Mu dI1681 insertion contained in each of the Tfr strains is found at a different site in the L. pneumophila chromosome (Mintz and Shuman 1987). Also, differences in .the direction of chromosome transfer may have affected the ability of individual Mu dI1681 Tfr donors to transfer selected markers. Our inability to detect cotransfer of the Thy and Trp phenotypes in matings between Mu dI 1681 Tfr donors and a Thy- Trp - recipient suggested that there was no detectable linkage between the thy and trp loci in strain Philadelphia- 1. Simon (1984) used the Tn5-Mob system to mobilize the chromosomes of E. coli and Rhizobium spp. In our study, a Tn5-Mob containing donor (CS293), in the presence of helper plasmid pRK24.4, transferred .the Trp and Gua phenotypes to auxotrophic recipients of strain Bloomington-2 (Table 2). Two other Tn5-Mob strains did not transfer these markers. The inability of these strains to transfer the Trp or Gua phenotypes could be explained by the location of Tn5-Mob relative to the trp or gua locus or by the direction
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of chromosome transfer in these strains. Currently, efforts are underway to introduce Tn5-Mob insertions into the Bloomington-2 chromosome to further characterize the chromosome mobilizing activity of Tn5-Mob in L. pneumophila. It is evident from our results that RK2::Mu plasmids and Tn5-Mob can be used to mobilize the L. pneumophila chromosome. Ultimately, we would like to use either or both of these systems t o construct a genetic map for L. pneumophila. However, to accomplish this, it will be necessary to identify additional genetic markers for L. pneumophila. As a result of the complex nutritional requirements of L. pneumophila and, consequently, the chemical composition of CAA medium, the types of auxotrophs that can be isolated from L. pneumophila are limited (Mintz et al. 1988). T o overcome this problem, we recently constructed donor and recipient strains that are resistant to a variety of antibiotics and amino acid analogues (C. Mintz, unpublished results). Therefore, as more genetic markers become available, it should be possible to use Tn5-Mob or Mu-mediated chromosome transfer to construct a rudimentary genetic map for L. pneumophila. Acknowledgements The authors thank Howard Shuman and David Figurski for helpful discussions, and Carleen Collins and Alan Kimura for critical review of the manuscript. The expert typing and graphic skills of Michelle Soto are gratefully acknowledged. Barrett, J.T., Rhodes, C.S., Ferber, D.M., et al. 1982. Construction of a genetic map for Caulobacter cresentus. J. Bacteriol. 149: 889-896. Beringer, J.E., Hoggan, S.A., and Johnston, A. W.B. 1978. Linkage mapping in Rhizobium leguminosarum by means of R plasmid-mediated recombination. J. Gen. Microbiol. 104: 20 1-207. Bittle, C., and Konopka, A. 1990. IncP-mediated transfer of loci involved with gas vesicle production in Ancylobacter aquaticus. J. Gen. Microbiol. 136: 1259-1263. Castilho, B., Olfson, P., and Casadaban, M.J. 1984. Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J. Bacteriol. 158: 488-495. Chatterjee, A.K., Ross, L.M., McEvoy, J.L., and Thurn, K.K. 1985. pULB 113, an RP4::mini-Mu plasmid, mediated chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia crysanthemi, and subspecies of Erwinia carotovora. Appl. Eviron. Microbiol. 50: 1-9. Chen, G.C., Lema, M., and Brown, A. 1984. Plasmid transfer into members of the family Legionellaceae. J. Infect. Dis. 150: 513-516. Chen, G.C., Brown, A., and Lema, M.W. 1986. Restriction endonuclease activities in the legionellae. Can. J. Microbiol. 32: 591-593. Deich, R.A., and Green, B.A. 1987. Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F ' factor. J. Bacteriol. 169: 1905-1910. Dreyfus, L.A. 1987. Virulence associated ingestion of Legionella pneumophila by HeLa cells. Microb. Pathog. 3: 45-52. Dreyfus, L.A., and Iglewski, B.H. 1985. Conjugation-mediated genetic exchange in Legionella pneumophila. J. Bacteriol. 161: 80-84. Engleberg, N.C., Cianciatto, N., Smith, J., and Eisenstein, B.I. 1988. Transfer and maintenance of small, mobilizable plasmids with ColEl replication origins in Legionella pneumophila. Plasmid, 20: 83-91.
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Fields, B.A., Barbaree, J.M., Shotts, E.B., et al. 1986. Comparison of guinea pig and protozoan models for determining the virulence of Legionella species. Infect. Immun. 53: 553-559. Figurski, D., Meyer, R., Miller, D.S., and Helinski, D.R. 1976. Generation of in vitro deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease. Gene, 1: 107-119. Forbes, K. J., and Perombelon, M.C.M. 1985. Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu. J. Bacteriol. 164: 1110-1 116. Hortwitz, M.A., and Silverstein, S.C. 1980. Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J . Clin. Invest. 66: 44.1-450. Ichige, A., Matsutani, S., Oishi, K., and Mizushima, S. 1989. Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain. J . Bacteriol. 171: 1825-1834. Keen, M.G., and Hoffman, P.S. 1985. Broad host range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome. J. Bacteriol. 162: 1331-1335. Lejeune, P., Mergeay, M., Van Gijsegem, F., et al. 1983. Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB 113(RP4: :Mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2. J. Bacteriol. 155: 1015-1026. Marra, A., and Shuman, H.A. 1989. Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J. Bacteriol. 171: 2238-2240. Meyer, R., Figurski, D., and Helinski, D.R. 1977. Physical and genetic studies with restriction endonucleases on the broad host range plasmid RK2. Mol. Gen. Genet. 152: 129-135. Mintz, C.S., and Shuman, H.A. 1987. Transposition of bacteriophage Mu in the legionnaires disease bacterium. Proc. Natl. Acad. Sci. U.S.A. 84: 4645-4649.
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Mintz, C.S., and Shuman, H.A. 1988. Genetics of Legionella pneumophila. Microbiol. Sci. 5: 292-295. Mintz, C.S., Chen, J., and Shuman, H.A. 1988. Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes. Infect. Immun. 56: 1449-1455. Oldham, L. J., and Rodgers, F.G. 1985. Adhesion, penetration and intracellular replication of Legionella pneumophila: an in vitro model of pathogenesis. J. Gen. Microbiol. 131: 697-706. Pischl, D.L., and Farrand, S.K. 1983. Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens. J . Bacteriol. 153: 1451-1460. Schoonejans, E., and Toussaint, A. 1983. Utilization of plasmid pULB113 (RP4::Mini-Mu) to construct a linkage map of Erwinia carotova subsp. chrysanthemi. J . Bacteriol. 154: 1489-1492. Silhavy, T. J., Berman, M.L., and Enquist, L. W. 1984. Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Simon, R. 1984. High frequency mobilization of gram negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol. Gen. Genet. 196: 413-420. Simon, R., Priefer, U., and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. BioTechniques, 1: 784-791. Towner, K. J. 1978. Chromosome mapping in Acinetobacter calcoaceticus J. Gen. Microbiol. 104: 175- 180. Van Gijsegem, F., and Toussaint, A. 1982. Chromosome transfer and R-prime formation by an RP4-mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Protease mirabilis. Plasmid, 7: 30-44.
