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Gene transfer in Legionella pneumophila
Microbes and Infection, 1, 1999, 1203−1209 © 1999 Éditions scientifiques et médicales Elsevier. All rights reserved
Gene transfer in Legionella pneumophila Clifford S. Mintz Bioinsights, Inc., 153 Dorchester Drive East, Windsor, NJ 08520, USA
ABSTRACT – This review describes the mechanisms of gene transfer in Legionella pneumophila. To date, conjugation and transformation have been reported for this organism. Recent reports indicate that an endogenous system of plasmid transfer appears to be required for the intracellular survival and multiplication of L. pneumophila in host cells. © 1999 Éditions scientifiques et médicales Elsevier gene transfer / Legionella pneumophila / molecular genetics / plasmids / conjugation
1. Introduction Legionella pneumophila, the causative agent of Legionnaires’ disease, is a Gram-negative facultative intracellular pathogen that can enter and grow within a variety of eukaryotic cells including human monocytes and alveolar macrophages [8, 9], cultured human and animal cells [2, 26] and free-living amoebae [4, 7, 24]. In contrast with many human pathogens, the natural habitat for L. pneumophila is fresh water aquatic environments where freeliving amoebae are thought to be its natural hosts [4, 28]. Intracellular growth of L. pneumophila in eukaryotic cells is dependent upon inhibition of host cell phagosomelysosome fusion [9]. Although inhibition of phagosomelysosome fusion is not unique to L. pneumophila, its ability to grow intracellularly in such a diverse array of eukaryotic cell types has intrigued both cell biologists and microbiologists since its initial isolation in 1976. This interest has led to the identification of a variety of genes that are required for entry and intracellular multiplication of L. pneumophila within host cells [16, 24, 30, 31, 32]. In contrast with the growing body of information regarding its intracellular lifestyle, much less is known about gene transfer in L. pneumophila. The purpose of this review is to describe the mechanisms of gene transfer in L. pneumophila and the role gene transfer may play in the intracellular survival and growth of this organism.
2. Mechanisms of genetic exhange in L. pneumophila The ability to exchange genetic information between different members of the same bacterial species is vital for continued survival of an organism in a given ecological niche. To date, three general mechanisms of genetic exchange have been described for bacteria: 1). transformation, 2). transduction, and 3). conjugation. Microbes and Infection 1999, 1203-1209
Transformation is the uptake of ‘naked’ DNA by competent bacterial recipients. Transduction involves transfer of DNA between bacteria by bacterial viruses (bacteriophages). In contrast, conjugation requires cell-to-cell contact between donors and recipients (mediated by sex pili) and subsequent transfer of DNA from donor to recipient cells. Further, the conjugation process is mediated by conjugative plasmids (carried by the donor) that carry genes that encode proteins required for mating pair formation and transfer of plasmid DNA from donor to recipient cells. In some instances, conjugative plasmids can integrate into the chromosome of donor cells to promote oriented transfer of chromosomal genes. Isogenic genomic DNA introduced into bacterial cells via transformation, transduction, or conjugation is subsequently recombined into the chromosomes of recipient cells by homologous recombination. In the mid-1980s, several groups [1, 3, 21] began to explore the mechanisms of genetic exchange utilized by L. pneumophila. Early transformation experiments in our laboratory, using untreated or calcium chloride-treated serogroup 1 strains of L. pneumophila, suggested that legionellae could not be transformed with plasmid DNA from the IncP, IncQ, IncF, or colE1 incompatibility groups. Our results, along with the results of others who performed similar transformation experiments, led to the notion that L. pneumophila was not capable of DNA transformation by artificial or natural means. However, several years after these early transformation experiments were performed, Andrea Marra in Howard Shuman’s laboratory demonstrated that L. pneumophila could be transformed with plasmid DNA via electroporation [16]. This discovery had a profound effect on the genetic analysis of L. pneumophila because, prior to this finding, genetic manipulation of this organism was very limited and extremely tedious. Although L. pneumophila was capable of transformation via electroporation, it was generally believed that L. pneumophila was not naturally competent for DNA trans1203
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formation. Surprisingly, Stone and Abu Kwaik [34] recently showed that L. pneumophila that produced type IV pili are naturally competent for DNA transformation. Serogroup 1 strains of L. pneumophila that produced type IV pili were easily transformed by isogenic chromosomal DNA and plasmid DNA that contained L. pneumophila DNA sequences. Interestingly, Stone and Abu Kwaik showed that transformation frequencies were reduced when competing DNA containing L. pneumophila DNA or vector DNA was added to transformation mixtures. On the basis of this finding, they speculated that, unlike other naturally transforming bacteria, uptake-specific sequences may not be involved in DNA uptake by L. pneumophila. Finally, Stone and Abu Kwaik showed that competence for DNA transformation was contingent upon expression of type IV pili, because a mutant that was defective for type IV pilus production could not be transformed by isogenic L. pneumophila DNA. The mutant regained competence for DNA transformation following complementation by wild-type type IV pili genes. Despite earlier contradictory reports, Stone and Abu Kwiak have provided convincing evidence that type IV pili-producing L. pneumophila are naturally competent for DNA transformation. It is important to note, however, that early transformation experiments were performed with serogroup 1 strains of L. pneumophila that did not produce type IV or an other detectable types of pili. Transduction has never been reported for L. pneumophila. Despite repeated attempts, we were unable to isolate or identify a bacteriophage(s) that uses L. pneumophila as a host [21]. Early on, we determined that L. pneumophila was insensitive to infection by bacteriophages P1, Lambda, Mu, and a variety of Pseudomonas aeruginosa phages. Further, attempts to isolate L. pneumophila specific phages from a variety of environmental sources including sewage, soil, and pond and stream water were unsuccessful. Finally, exposure of legionellae to ultraviolet light or ethidium bromide (to induce the lytic cycle of Legionella -specific prophages) failed to yield any detectable phage particles in cell lysates. The first report of plasmid transfer into L. pneumophila was published in 1984 by Arnold Brown and his colleagues [1]. They showed that broad host range conjugative plasmids of the IncP1 incompatibility group, e.g., RP4 could be transferred from Escherichia coli to L. pneumophila strain Bloomington 2 (serogroup 3) with a frequency of 10–3 transconjugants per recipient. Moreover, L. pneumophila transconjugants that harbored RP4 transferred the plasmid with moderate frequency to a restrictionmodification strain of E. coli. However, Bloomington 2 transconjugants failed to transfer RP4 via conjugation to other strains of L. pneumophila. In 1985, Lawrence Dreyfus and Barbara Iglewski [3] demonstrated that broad host range plasmids RP1 and R68.45 (IncP1), S-a (IncW) and R40a (IncC) could be transferred by conjugation from E. coli to L. pneumophila strain Knoxville (serogroup 1) at frequencies ranging from 10–3 to 10–5 per recipient. Further, they showed that RP1 and R68.45 could be transferred from strain Knoxville to other serogroup 1 strains and two different Legionella species. Finally, mating experiments between a wild-type 1204
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Table I. Plasmid transfer in L. pneumophila. Name RK2 R68.45 RP1 RP4 S-a R40a RSF1010 F colE1
Incompatibility group P P P P W C Q F1 ---
Transfer frequency (per recipient)
Reference
5.0 × 10–1 – 3.0 × 10–6 4.7 × 10–3 6.6 × 10–3 5.0 × 10–3 2.2 × 10–4 5.4 × 10–5 10–3 – 10–6 6.4 × 10–4– 1.5 × 10–3 10–4 – 10–5
[21] [3] [3] [1] [3] [3] [32, 36] [37] [5]
strain of Knoxville 1 that harbored R68.45 and a Knoxville 1 thymidine auxotroph revealed that R68.45 could mobilize the L. pneumophila chromosome to repair the thymidine defect in recipient cells. Unfortunately, R68.45mediated transfer of the thy locus occurred at extremely low frequencies, ca 10–9 per recipient [3]. Nevertheless, this was the first report to clearly document that conjugation, mediated by broad host range plasmids, was a viable mechanism for genetic exchange in L. pneumophila. To date, a variety of plasmids from different incompatibility groups have been introduced by conjugation into L. pneumophila (table I). Several of these plasmids can be mobilized (IncQ, colE 1) or are self transmissible by conjugation (IncP, IncN, IncW) between different serogroups of L. pneumophila. Of interest, plasmids of the IncP1 and IncQ incompatibility groups can be maintained in L. pneumophila in the absence of selective antibiotics [21], whereas colE1 plasmids and F are rapidly lost without selection [5, 37].
3. Chromosome mobilization studies Although plasmids of varying sizes had been described for L. pneumophila prior to 1985 [11, 12, 14, 18, 25], early reports suggested that these plasmids were cryptic and nonconjugative. These findings prompted many investigators to presume that L. pneumophila lacked an endogenous system of chromosome transfer. Consequently, it was generally believed that an artificial system of chromosome transfer would have to be constructed to conduct a genetic analysis of L. pneumophila. In the late 1980s, work in my laboratory was focused on developing a conjugation-based chromosomal gene transfer system that could be used to identify virulence genes in L. pneumophila. At that time, it was well known that IncP plasmids could mediate the transfer of chromosomal genes of Gram-negative bacteria that lack endogenous systems of chromosome transfer. Moreover, the work of Dreyfus and Iglewski suggested that IncP plasmids could mediate the exchange of chromosomal markers in L. pneumophila. In light of these observations, we began to evaluate the use of IncP plasmids to mobilize the L. pneumophila chromosome. Initial experiments using native IncP plasmids like RK2 to mobilize the L. pneumophila chromosome were unsucMicrobes and Infection 1999, 1203-1209
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Table II. Tfr- and Mu-mediated mobilization of chromosomal markers in L. pneumophilaa. Donor RK2 RK2::MudXc RK2::MudI1681d Tfr-1e Tfr-2e RK2 RK2:MudI1681 Tfr-1 Tfr-2
Selected marker
Recombination frequencyb
Thy+ Thy+ Thy+ Thy+ Thy+ Trp+ Trp+ Trp+ Trp+
6.0 × 10–8 1.4 × 10–8 6.8 × 10–6 2.2 × 10–7 8.8 × 10–8 2.5 × 10–8 2.6 × 10–7 3.9 × 10–6 1.7 × 10–6
a Donor and recipients used in these mating experiments were all derived from strain Philadelphia 1. Mating experiments were performed according to the methods described by Mintz and Zou [23]. bNumber of Thy+ or Trp+ exconjugants divided by the total number of recipients. cMudX is a mini-Mu phage that is defective for replicative transposition. dMu dI1681 is a mini-Mu transcriptional fusion phage that is capable of replicative transposition. e Tfr strains contain RK2:MudI1681 and MudI1681 chromosomal insertion.
cessful. However, at the time, several investigators had reported that IncP plasmids that contained bacteriophage Mu insertions exhibited an increased capacity (as compared with native plasmids) to promote the transfer of chromosomal genes in Gram-negative bacteria [6, 12]. Chromosome mobilization in donors that harbored Mu-containing plasmids resulted from replicon fusion (between the plasmid and host chromosome) that occurred during replicative transposition of bacteriophage Mu [12]. In a previous study [19], we showed that Mu was capable of replicative transposition in serogroup 1 strains of L. pneumophila. This suggested that it may be possible to use Mu-containing IncP plasmids to mobilize the L. pneumophila chromosome. To that end, we performed mating experiments between prototrophic RK2::Mucontaining serogroup 1 donors and several auxotrophic recipients. of L. pneumophila. The tryptophan (trp) and thymidine (thy) auxotrophs used in these experiments were isolated using selection protocols and a semi-defined medium (called CAA) that we had previously developed [20]. The results of these mating experiments clearly demonstrated that Mu-containing IncP1 plasmids could promote the transfer of chromosomal markers in strains Philadelphia 1 (table II) and Bloomington 2 [21, 23]. The absence of chromosome transfer in matings with donors that harbored RK2 or RK2 that contained MudX (a replication defective mutant of Mu) indicated that chromosome mobilization probably resulted from a mechanism involving Mu-mediated replicon fusion. Also, heterospecific matings between L. pneumophila Trp+ and Thy+ exconjugants and trp and thy auxotrophs of E. coli showed that complementation by R prime plasmids was not responsible for the Trp+ and Thy+ phenotypes of the exconjugants. One disadvantage of using Mu-containing plasmids for chromosome mobilization is that transfer of chromosomal markers usually does not occur in a polar, oriented fashion from a single fixed origin of transfer. Instead, chromosome Microbes and Infection 1999, 1203-1209
transfer usually proceeds from multiple origins in different donors, which makes co-transfer and linkage experiments difficult to interpret. To overcome this potential problem, we sought to use transposon-facilitated recombination (Tfr) to mobilize the L. pneumophila chromosome. With Tfr [27], regions of DNA sequence homology are created between a conjugative plasmid and the donor chromosome by introducing identical transposable elements into each of the replicons (figure 1). Integration of the plasmid via homologous recombination at the site of the insertion in the donor chromosome is thought to promote an oriented transfer of chromosomal markers using the integration site as the sole origin of chromosome transfer. In a previous study [19], we introduced more than 25 independent Mu insertions into the Philadelphia 1 chromosome using the mini-Mu fusion phage MudI1681. Southern hybridization experiments confirmed that each of the insertions mapped to a different region of the Philadelphia 1 chromosome. Further, several strains that contained MudI1681 chromosomal insertions also harbored the plasmid that was originally used to introduce MudI1681 into strain Philadelphia 1. This provided us with an ideal opportunity to determine whether Tfr could be used to promote oriented transfer of chromosomal markers in L. pneumophila. To test this, we performed mating experiments with several of these MudI1681containing strains as donors and a double auxotrophic mutant (trp and thy) as a recipient [23]. The results of these experiments showed that Tfr donors transferred the trp locus at a higher frequency than donors that harbored Mu-containing plasmids alone ([23], table II). Transfer of the thy locus varied between donors [23]. Unfortunately, we never observed co-transfer of the Thy+ and Trp+ markers during Tfr mating experiments. The lack of linkage between the thy and trp loci indicated that it would be necessary to create additional double and triple auxotrophic strains to determine whether Tfr could mediate the oriented transfer of chromosomal markers in L. pneumophila. Nevertheless, our results indicated that Tfr can be used to promote transfer of chromosomal genes in L. pneumophila and that the thy and trp loci were not in close proximity to one another on the L. pneumophila chromosome. In the early 1980s, Simon [33] constructed the Tn5Mob transposon that contains the Mob region of the IncP plasmid RP4. He reported that Gram-negative bacteria that contained Tn5-Mob chromosomal insertions and a helper IncP plasmid (to supply necessary transfer proteins in trans) could transfer chromosomal markers in a polar, oriented fashion during conjugation (figure 2). The Tn5Mob system offered an advantage over Mu-based Tfr because, unlike Mu, Tn5 is not capable of secondary transposition in L. pneumophila. The inability of Tn5-Mob to undergo secondary transposition in L. pneumophila insured that chromosomal gene transfer in Tn5-Mobcontaining donors occurred from a single fixed origin of transfer (rather than from multiple origins) during conjugation. To test this, we introduced several Tn5-Mob insertions in strain Philadelphia 1 that mapped to different regions of the L. pneumophila chromosome [23]. Following intro1205
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Figure 1. Schematic representation of transposon-facilitated recombination 1. Regions of homology are created in the donor by Mu insertions found in the self-transmissible IncP plasmid and L. pneumophila chromosome. 2. Homologous recombination between plasmid and chromosomal Mu insertions. 3. IncP plasmid is integrated into the L. pneumophila chromosome during recombination. 4. The origin of plasmid transfer (oriT) of the integrated plasmid serves as the origin of chromosome transfer which results in the transfer of chromosomal genes from donor to recipients during conjugation.
duction of a helper IncP plasmid, mating experiments were performed between Tn5-Mob donors and several auxotrophic recipients, e.g., guanine (gua), trp, and thy,. Although Tn5-Mob donors transferred each of the abovementioned chromosomal markers, recombination frequencies in recipients were 10-fold lower than those obtained with the Mu-based Tfr system [23]. The reason for differences in the recombination frequencies of selected markers between the Mu-based Tfr and Tn5-Mob systems is not clear. It is possible that the direction of chromosome transfer or location of Tn5-Mob insertions relative to the selected markers could account for the observed differences. Nonetheless, our results clearly demonstrated that three different chromosome mobilization systems could be employed to develop a conjugationbased system of gene transfer for L. pneumophila.
4. Identification of indigenous conjugative plasmids As mentioned above, a number of indigenous plasmids ranging in size from 35 to 128 kb have been identified in L. pneumophila (table III). Many of these plasmids were originally identified in serogroup 1 strains [10, 14, 18]. This led several investigators to speculate that virulence 1206
genes may be carried on the plasmids, because the majority of L. pneumophila isolates from patients with Legionnaires’ disease were from serogroup 1. However, since most serogroup 1 clinical isolates did not contain plasmids, it was generally assumed that plasmids were not required for virulence. Consequently, these plasmids were not studied in much detail following their identification. Several groups had reported that a number of environmental and clinical serogroup 1 strains harbored a 128-kb plasmid [10, 14]. This observation, along with our previous work which showed that conjugation was possible in L. pneumophila, led us to investigate the conjugative properties of the 128-kb plasmid, which we called pCH1. Mating experiments between legionellae that harbored pCH1 and plasmidless recipients revealed that pCH1 was self-transmissible by conjugation to several serogroup 1 strains of L. pneumophila. Plasmid transfer frequencies ranged from 10–3 to 10–4 per recipient [22]. Interestingly, pCH1 could not be transferred by conjugation from serogroup 1 donors to serogroup 3 strains or E. coli. Additional mating experiments between legionellae that harbored pCH1 and several auxotrophic recipients (serogroups 1 and 3) showed that pCH1 did not promote transfer of three chromosomal markers (gua, thy or trp). Despite numerous attempts, we were unable to identify a phenotype conferred upon strains that harbored pCH1. Microbes and Infection 1999, 1203-1209
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Figure 2. Tn5-Mob mediated mobilization of the L. pneumophila chromosome. 1. Tn5-Mob transposon is introduced into L. pneumophila on a suicide vector. 2. Tn5-Mob transposes from the suicide vector into the L. pneumophila chromosome. 3. A helper IncP plasmid is transferred into the donor that contains a Tn5-Mob chromosomal insertion. The helper IncP plasmid supplies all of the necessary transfer functions (in trans) that are required for chromosome mobilization and gene transfer. 4. Mob sequences contained in Tn5-Mob transposon act as the origin of chromosome transfer to transfer chromosomal markers during conjugation.
In a similar study, Tully [35] showed that a 55-kb plasmid found in a clinical serogroup 1 strain called Dodge was self transmissible by conjugation to other serogroup 1 isolates. Like pCH1, this plasmid could not be transferred by conjugation from L. pneumophila to E. coli or P. aeruginosa. The plasmid was compatible with plasmids of the IncP and IncW incompatibility groups. Further, strains that harbored the plasmid exhibited increased resistance to killing by ultraviolet light as compared with isogenic plasmidless isolates. Another serogroup 1 plasmid, designated pLPG36, was studied in great detail by Lopez de Felipe [13]. He demonstrated that one of four recombinant plasmids
constructed from pLPG36 in pUC18 could be mobilized by IncP plasmids. Unfortunately, Lopez de Felipe failed to determine whether the native plasmid, pLPG36, was self transmissible by conjugation. Again, as was the case with pCH1, there was no obvious phenotype associated with pLPG36. Nevertheless, the results from our work and that of Tully indicated that serogroup 1 strains of L. pneumophila harbor indigenous plasmids that are self transmissible by conjugation. Furthermore, it is possible that integration of indigenous plasmids into the L. pneumophila chromosome may promote the transfer of chromosomal genes during conjugation.
Table III. Indigenous plasmids found in L. pneumophila. Designation pCH1 pLP3 pUH2 pLPG36 Dodge pUPH1 pLp4269
Size (kb)
Conjugative
Phenotype
Sourcea
Reference
128 92 69 58 55 50 37.5
+ ND ND ND + ND ND
cryptic cryptic ND cryptic UV resistance ND ND
E/C C E E/C C E/C E/C
[22] [18] [14] [13] [35] [15] [14]
a
Bacteria that harbored plasmids were isolated from either clinical or environmental samples. C, clinical; E, environmental; ND, not determined.
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5. Conjugation and intracellular growth The identification of genes that permit L. pneumophila to survive and grow intracellularly within eukaryotic host cells has been a subject of intense research over the past five years. To date, two groups (Howard Shuman’s at Columbia University and Ralph Isberg’s at Tufts University), have identified 23 genes located in two unlinked regions of the L. pneumophila chromosome that are necessary for the intracellular growth of this organism. The Shuman group calls these genes icm (for ‘intracellular multiplication’), whereas the Isberg group calls them dot (for ‘defect in organelle trafficking’). All 23 genes have been cloned and their DNA sequences determined [16, 29, 30–32]. Sequence analysis of proteins encoded by icm genes revealed that four of the Icm proteins (IcmP, IcmO, IcmL and IcmE) contained substantial amino acid similarity to plasmid-encoded genes involved in DNA transfer [30–32, 36]. An additional protein (DotB) was found to be homologous to a large family of nucleotide-binding proteins that include members of various conjugal transfer systems [36]. This suggested that an endogenous conjugation system may exist in L. pneumophila and that wild-type strains of L. pneumophila may be able to mediate plasmid DNA transfer. To test this, the Shuman and Isberg groups introduced derivatives of the non-self-transmissible plasmid IncQ plasmid RSF1010 into several wild-type donors and performed mating experiments with appropriately marked antibiotic-resistant recipients. Both groups found that RSF1010 could be easily transferred via conjugation [32, 36]. Further, they showed that conjugation was dependent on certain icm/dot genes, because mutations in these genes interfered with conjugation [32]. Of interest, in a recent report, Segal and Shuman [31] showed that a functional RSF1010 mobilization system in wild-type strains of L. pneumophila was required for intracellular growth in macrophage-like cells. Both groups [31, 36] have proposed that the icm/dot conjugation system plays a key role in the intracellular growth of L. pneumophila by transporting an effector molecule(s) into host cells that inhibits phagosome-lysosome fusion. This, in turn, allows L. pneumophila to multiply unabated within unfused phagosomes. The natural substrate for icm/dot system is likely to be a protein, rather than DNA, because inhibition of phagosome-lysosome fusion occurs within 30 minutes following ingestion of legionellae and there is probably not enough time for DNA to be transferred and expressed within this time period [31].
6. Concluding remarks It is evident from this review that our understanding of the mechanisms of gene transfer in L. pneumophila has increased dramatically in recent years. For example, we now know that L. pneumophila 1) is capable of natural DNA transformation and can be transformed by electroporation, 2) harbors indigenous conjugative plasmids and 3) possesses a chromosome-based, endogenous system of 1208
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plasmid transfer. Nevertheless, additional research will be required to determine whether L. pneumophila utilizes one or more of these mechanisms to promote gene transfer in nature. The advent of bacterial genome sequencing has rendered conventional methods of gene mapping obsolete. Therefore, it is unlikely that transformation or conjugation will be considered when constructing a genetic and physical map of the L. pneumophila chromosome. Nevertheless, identification of the icm/dot system of plasmid transfer raises some important fundamental questions regarding gene transfer in L. pneumophila. First, can plasmids other than those belonging to the IncQ incompatibility group be mobilized by the icm/dot system? Second, are indigenous conjugative plasmids dependent on icm/dot or truly self transmissible by conjugation? Finally can the icm/dot system promote the transfer of chromosomal genes in L. pneumophila? Answers to these questions will undoubtedly enhance our understanding of the genetic organization and underlying pathogenic mechanisms of this intriguing intracellular pathogen.
Acknowledgments I want to thank Chang Hua Zou for excellent technical assistance and her dedication to the study of gene transfer in L. pneumophila. The artistic talents of Vincent Racaniello and the helpful and insightful discussions with Dave Figurski are greatly appreciated and acknowledged. Finally, I want to thank Howard Shuman for teaching me everything I ever wanted to know about bacterial genetics and then some.
References [1] Chen G.C.C., Lema M., Brown A., Plasmid transfer into members of the family Legionellaceae, J. Infect. Dis. 150 (1984) 513–516. [2] Dreyfus L.A., Virulence associated ingestion of Legionella pneumophila by HeLa cells, Microb. Pathogen. 3 (1987) 45–52. [3] Dreyfus L.A., Iglewski B.H., Conjugation mediated genetic exchange in L, pneumophila, J. Bacteriol. 161 (1985) 80–84. [4] Fields B.S., Barbaree J.M., Shotts E.B., Feeley J.C., Morrill W., Sanden G.S., Dykstra M.J., Comparison of the guinea pig and protozoan models for determining the virulence of Legionella species, Infect. Immun. 53 (1986) 553–559. [5] Engleberg N.C., Cianciatto N., Smith J., Eisenstein B.I., Transfer and maintenance of small mobilizable plasmids with ColE1 replication origins in Legionella pneumophila, Plasmid 20 (1988) 83–91. [6] Forbes K.J., Perombelon M.C.M., Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu, J. Bacteriol. 164 (1985) 1110–1116. [7] Holden E.P., Winkler H.H., Wood D.O., Leinbach E.D., Intracellular growth of Legionella pneumophila with Acanthamoeba castellani Neff, Infect. Immun. 45 (1984) 18–24. Microbes and Infection 1999, 1203-1209
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[8] Horowitz M.A., Silverstein S.C., Legionnaires disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes, J. Clin. Invest. 66 (1980) 441–450. [9] Horowitz M.A., The Legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes, J. Exp. Med. 158 (1983) 2108–2126. [10] Johnson S.R., Schalla W.O., Plasmids of serogroup 1 strains of Legionella pneumophila, Cur. Microbiol. 7 (1982) 143–146. [11] Knudson G.B., Mikesell P., A plasmid in Legionella pneumophila, Infect. Immun. 29 (1980) 1092–1095. [12] Lejune P., Mergeay M., VanGijsegem F., Chromosome transfer and R-prime plasmid formation mediated by pULB113 (RP4:Mini-Mu) in Alcaligenes eutrophus a CH34 and Pseudomonas fluorescens 6.2, J. Bacteriol. 155 (1983) 1015–1026. [13] Lopez D.E Felipe F., Cloning, mapping and conjugal mobility of PLPG36, a common plasmid from Legionella pneumophila serogroup-1, J. Gen. Micro. 139 (1993) 3171–3175. [14] Maher W.E., Plouffe J.F., Para M.F., Plasmid profiles of clinical and environmental isolates of Legionella pneumophila serogroup 1, J. Clin. Micro. (1983) 1422–1423. [15] Mansfield S.D., Marrie T.J., Bezanson G.S., Characterization and cloning of a 37.6 kb plasmid carried by Legionella pneumophila recovered from patients and hospital water over a 12-year period, Can. J. Microbiol. 43 (1997) 193–197. [16] Marra A., Blander S.J., Horwitz M.A., Shuman H.A., Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages, Proc. Natl. Acad. Sci. USA 89 (1992) 9607–9611. [17] Mellado A., Aguero J., Seoane A., Rodriquez-Solorzano L.M., Plasmid profiles of Legionella pneumophila strains isolated in Spain, Eur. J. Epidemiol. 2 (1986) 63–66. [18] Mikesell P., Ezzell J.W., Knudson G.B., Isolation of plasmids in Legionella pneumophila and Legionella-like organisms, Infect. Immun. 37 (1981) 1270–1272. [19] Mintz C.S., Shuman H.A., Transposition of bacteriophage Mu in the Legionnaires’ disease bacterium, Proc. Natl. Acad. Sci. USA 84 (1987) 4645–4649. [20] Mintz C.S., Chen J., Shuman H.A., Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes, Infect. Immun. 56 (1988) 1449–1455. [21] Mintz C.S., Shuman H.A., Genetics of Legionella pneumophila, Microbiol. Sci. 5 (1988) 292–295. [22] Mintz C.S., Fields B.S., Zou C.H., Isolation and characterization of a conjugative plasmid from Legionella pneumophila, J. Gen. Micro. 138 (1992) 1379–1386.
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[23] Mintz C.S., Zou C.H., Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob, Can. J. Micro. 38 (1992) 664–671. [24] Newsome A.L., Baker R.L., Miller R.D., Arnold R.R., Interactions between Naeglaria fowleri and Legionella pneumophila, Infect. Immun. 50 (1985) 449–452. [25] Nolte F.S., Conlin C.A., Roisin A.J., Redmond S.R., Plasmids as epidemiological markers in nosocomial Legionnaires’ disease, J. Infect. Dis. 149 (1984) 251–256. [26] Pearlman D., Jiwa A.H., Engleberg N.C., Eisenstein B.I., Growth of Legionella pneumophila in a human macrophagelike (U937) cell line, Microb. Pathogen. 5 (1988) 87–95. [27] Pischl D.L., Farrand S.K., Transposon-facilitated chromosome mobilization in Agrobacterium tumemfaciens, J. Bacteriol. 153 (1983) 1451–1460. [28] Rowbotham T.J., Current views on the relationship between amoebae, legionellae and man, Israel. J. of Med. Sci. 22 (1986) 678–689. [29] Roy C.R., Berger K.H., Isberg R.R., Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur with minutes of bacterial uptake, Mol. Micro. 28 (1998) 663–674. [30] Segal G., Shuman H.A., Characterization of a new region required for macrophage killing by Legionella pneumophila, Infect. Immun. 65 (1997) 5057–5066. [31] Segal G., Shuman H.A., Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of the IncQ plasmid RSF1010, Mol. Micro. 30 (1998) 197–208. [32] Segal G., Purcell M., Shuman H.A., Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome, Proc. Natl. Acad. Sci. USA 95 (1998) 1669–1674. [33] Simon R., High frequency mobilization of gram negative replicons by the in vitro constructed Tn5-Mob transposon, Mol. Gen. Genet. 196 (1994) 413–420. [34] Stone B.J., Abu Kwiak Y., Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili, J. Bacteriol. 181 (1999) 1395–1402. [35] Tully M., A plasmid from a virulent strain of Legionella pneumophila is conjugative and confers resistance to ultraviolet light, FEMS Microb. Lett. 90 (1991) 43–48. [36] Vogel J.P., Andrews H.L., Wong S.K., Isberg R.R., Conjugative transfer by the virulence system of Legionella pneumophila, Science 279 (1998) 873–876. [37] Wiater L.A., Marra A., Shuman H.A., Escherichia coli F plasmid transfers to and replicates within Legionella pneumophila: an alternative to using an RP4-based system for gene delivery, Plasmid 32 (1994) 280–294.
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Gene transfer in Legionella pneumophila Clifford S. Mintz Bioinsights, Inc., 153 Dorchester Drive East, Windsor, NJ 08520, USA
ABSTRACT – This review describes the mechanisms of gene transfer in Legionella pneumophila. To date, conjugation and transformation have been reported for this organism. Recent reports indicate that an endogenous system of plasmid transfer appears to be required for the intracellular survival and multiplication of L. pneumophila in host cells. © 1999 Éditions scientifiques et médicales Elsevier gene transfer / Legionella pneumophila / molecular genetics / plasmids / conjugation
1. Introduction Legionella pneumophila, the causative agent of Legionnaires’ disease, is a Gram-negative facultative intracellular pathogen that can enter and grow within a variety of eukaryotic cells including human monocytes and alveolar macrophages [8, 9], cultured human and animal cells [2, 26] and free-living amoebae [4, 7, 24]. In contrast with many human pathogens, the natural habitat for L. pneumophila is fresh water aquatic environments where freeliving amoebae are thought to be its natural hosts [4, 28]. Intracellular growth of L. pneumophila in eukaryotic cells is dependent upon inhibition of host cell phagosomelysosome fusion [9]. Although inhibition of phagosomelysosome fusion is not unique to L. pneumophila, its ability to grow intracellularly in such a diverse array of eukaryotic cell types has intrigued both cell biologists and microbiologists since its initial isolation in 1976. This interest has led to the identification of a variety of genes that are required for entry and intracellular multiplication of L. pneumophila within host cells [16, 24, 30, 31, 32]. In contrast with the growing body of information regarding its intracellular lifestyle, much less is known about gene transfer in L. pneumophila. The purpose of this review is to describe the mechanisms of gene transfer in L. pneumophila and the role gene transfer may play in the intracellular survival and growth of this organism.
2. Mechanisms of genetic exhange in L. pneumophila The ability to exchange genetic information between different members of the same bacterial species is vital for continued survival of an organism in a given ecological niche. To date, three general mechanisms of genetic exchange have been described for bacteria: 1). transformation, 2). transduction, and 3). conjugation. Microbes and Infection 1999, 1203-1209
Transformation is the uptake of ‘naked’ DNA by competent bacterial recipients. Transduction involves transfer of DNA between bacteria by bacterial viruses (bacteriophages). In contrast, conjugation requires cell-to-cell contact between donors and recipients (mediated by sex pili) and subsequent transfer of DNA from donor to recipient cells. Further, the conjugation process is mediated by conjugative plasmids (carried by the donor) that carry genes that encode proteins required for mating pair formation and transfer of plasmid DNA from donor to recipient cells. In some instances, conjugative plasmids can integrate into the chromosome of donor cells to promote oriented transfer of chromosomal genes. Isogenic genomic DNA introduced into bacterial cells via transformation, transduction, or conjugation is subsequently recombined into the chromosomes of recipient cells by homologous recombination. In the mid-1980s, several groups [1, 3, 21] began to explore the mechanisms of genetic exchange utilized by L. pneumophila. Early transformation experiments in our laboratory, using untreated or calcium chloride-treated serogroup 1 strains of L. pneumophila, suggested that legionellae could not be transformed with plasmid DNA from the IncP, IncQ, IncF, or colE1 incompatibility groups. Our results, along with the results of others who performed similar transformation experiments, led to the notion that L. pneumophila was not capable of DNA transformation by artificial or natural means. However, several years after these early transformation experiments were performed, Andrea Marra in Howard Shuman’s laboratory demonstrated that L. pneumophila could be transformed with plasmid DNA via electroporation [16]. This discovery had a profound effect on the genetic analysis of L. pneumophila because, prior to this finding, genetic manipulation of this organism was very limited and extremely tedious. Although L. pneumophila was capable of transformation via electroporation, it was generally believed that L. pneumophila was not naturally competent for DNA trans1203
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formation. Surprisingly, Stone and Abu Kwaik [34] recently showed that L. pneumophila that produced type IV pili are naturally competent for DNA transformation. Serogroup 1 strains of L. pneumophila that produced type IV pili were easily transformed by isogenic chromosomal DNA and plasmid DNA that contained L. pneumophila DNA sequences. Interestingly, Stone and Abu Kwaik showed that transformation frequencies were reduced when competing DNA containing L. pneumophila DNA or vector DNA was added to transformation mixtures. On the basis of this finding, they speculated that, unlike other naturally transforming bacteria, uptake-specific sequences may not be involved in DNA uptake by L. pneumophila. Finally, Stone and Abu Kwaik showed that competence for DNA transformation was contingent upon expression of type IV pili, because a mutant that was defective for type IV pilus production could not be transformed by isogenic L. pneumophila DNA. The mutant regained competence for DNA transformation following complementation by wild-type type IV pili genes. Despite earlier contradictory reports, Stone and Abu Kwiak have provided convincing evidence that type IV pili-producing L. pneumophila are naturally competent for DNA transformation. It is important to note, however, that early transformation experiments were performed with serogroup 1 strains of L. pneumophila that did not produce type IV or an other detectable types of pili. Transduction has never been reported for L. pneumophila. Despite repeated attempts, we were unable to isolate or identify a bacteriophage(s) that uses L. pneumophila as a host [21]. Early on, we determined that L. pneumophila was insensitive to infection by bacteriophages P1, Lambda, Mu, and a variety of Pseudomonas aeruginosa phages. Further, attempts to isolate L. pneumophila specific phages from a variety of environmental sources including sewage, soil, and pond and stream water were unsuccessful. Finally, exposure of legionellae to ultraviolet light or ethidium bromide (to induce the lytic cycle of Legionella -specific prophages) failed to yield any detectable phage particles in cell lysates. The first report of plasmid transfer into L. pneumophila was published in 1984 by Arnold Brown and his colleagues [1]. They showed that broad host range conjugative plasmids of the IncP1 incompatibility group, e.g., RP4 could be transferred from Escherichia coli to L. pneumophila strain Bloomington 2 (serogroup 3) with a frequency of 10–3 transconjugants per recipient. Moreover, L. pneumophila transconjugants that harbored RP4 transferred the plasmid with moderate frequency to a restrictionmodification strain of E. coli. However, Bloomington 2 transconjugants failed to transfer RP4 via conjugation to other strains of L. pneumophila. In 1985, Lawrence Dreyfus and Barbara Iglewski [3] demonstrated that broad host range plasmids RP1 and R68.45 (IncP1), S-a (IncW) and R40a (IncC) could be transferred by conjugation from E. coli to L. pneumophila strain Knoxville (serogroup 1) at frequencies ranging from 10–3 to 10–5 per recipient. Further, they showed that RP1 and R68.45 could be transferred from strain Knoxville to other serogroup 1 strains and two different Legionella species. Finally, mating experiments between a wild-type 1204
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Table I. Plasmid transfer in L. pneumophila. Name RK2 R68.45 RP1 RP4 S-a R40a RSF1010 F colE1
Incompatibility group P P P P W C Q F1 ---
Transfer frequency (per recipient)
Reference
5.0 × 10–1 – 3.0 × 10–6 4.7 × 10–3 6.6 × 10–3 5.0 × 10–3 2.2 × 10–4 5.4 × 10–5 10–3 – 10–6 6.4 × 10–4– 1.5 × 10–3 10–4 – 10–5
[21] [3] [3] [1] [3] [3] [32, 36] [37] [5]
strain of Knoxville 1 that harbored R68.45 and a Knoxville 1 thymidine auxotroph revealed that R68.45 could mobilize the L. pneumophila chromosome to repair the thymidine defect in recipient cells. Unfortunately, R68.45mediated transfer of the thy locus occurred at extremely low frequencies, ca 10–9 per recipient [3]. Nevertheless, this was the first report to clearly document that conjugation, mediated by broad host range plasmids, was a viable mechanism for genetic exchange in L. pneumophila. To date, a variety of plasmids from different incompatibility groups have been introduced by conjugation into L. pneumophila (table I). Several of these plasmids can be mobilized (IncQ, colE 1) or are self transmissible by conjugation (IncP, IncN, IncW) between different serogroups of L. pneumophila. Of interest, plasmids of the IncP1 and IncQ incompatibility groups can be maintained in L. pneumophila in the absence of selective antibiotics [21], whereas colE1 plasmids and F are rapidly lost without selection [5, 37].
3. Chromosome mobilization studies Although plasmids of varying sizes had been described for L. pneumophila prior to 1985 [11, 12, 14, 18, 25], early reports suggested that these plasmids were cryptic and nonconjugative. These findings prompted many investigators to presume that L. pneumophila lacked an endogenous system of chromosome transfer. Consequently, it was generally believed that an artificial system of chromosome transfer would have to be constructed to conduct a genetic analysis of L. pneumophila. In the late 1980s, work in my laboratory was focused on developing a conjugation-based chromosomal gene transfer system that could be used to identify virulence genes in L. pneumophila. At that time, it was well known that IncP plasmids could mediate the transfer of chromosomal genes of Gram-negative bacteria that lack endogenous systems of chromosome transfer. Moreover, the work of Dreyfus and Iglewski suggested that IncP plasmids could mediate the exchange of chromosomal markers in L. pneumophila. In light of these observations, we began to evaluate the use of IncP plasmids to mobilize the L. pneumophila chromosome. Initial experiments using native IncP plasmids like RK2 to mobilize the L. pneumophila chromosome were unsucMicrobes and Infection 1999, 1203-1209
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Table II. Tfr- and Mu-mediated mobilization of chromosomal markers in L. pneumophilaa. Donor RK2 RK2::MudXc RK2::MudI1681d Tfr-1e Tfr-2e RK2 RK2:MudI1681 Tfr-1 Tfr-2
Selected marker
Recombination frequencyb
Thy+ Thy+ Thy+ Thy+ Thy+ Trp+ Trp+ Trp+ Trp+
6.0 × 10–8 1.4 × 10–8 6.8 × 10–6 2.2 × 10–7 8.8 × 10–8 2.5 × 10–8 2.6 × 10–7 3.9 × 10–6 1.7 × 10–6
a Donor and recipients used in these mating experiments were all derived from strain Philadelphia 1. Mating experiments were performed according to the methods described by Mintz and Zou [23]. bNumber of Thy+ or Trp+ exconjugants divided by the total number of recipients. cMudX is a mini-Mu phage that is defective for replicative transposition. dMu dI1681 is a mini-Mu transcriptional fusion phage that is capable of replicative transposition. e Tfr strains contain RK2:MudI1681 and MudI1681 chromosomal insertion.
cessful. However, at the time, several investigators had reported that IncP plasmids that contained bacteriophage Mu insertions exhibited an increased capacity (as compared with native plasmids) to promote the transfer of chromosomal genes in Gram-negative bacteria [6, 12]. Chromosome mobilization in donors that harbored Mu-containing plasmids resulted from replicon fusion (between the plasmid and host chromosome) that occurred during replicative transposition of bacteriophage Mu [12]. In a previous study [19], we showed that Mu was capable of replicative transposition in serogroup 1 strains of L. pneumophila. This suggested that it may be possible to use Mu-containing IncP plasmids to mobilize the L. pneumophila chromosome. To that end, we performed mating experiments between prototrophic RK2::Mucontaining serogroup 1 donors and several auxotrophic recipients. of L. pneumophila. The tryptophan (trp) and thymidine (thy) auxotrophs used in these experiments were isolated using selection protocols and a semi-defined medium (called CAA) that we had previously developed [20]. The results of these mating experiments clearly demonstrated that Mu-containing IncP1 plasmids could promote the transfer of chromosomal markers in strains Philadelphia 1 (table II) and Bloomington 2 [21, 23]. The absence of chromosome transfer in matings with donors that harbored RK2 or RK2 that contained MudX (a replication defective mutant of Mu) indicated that chromosome mobilization probably resulted from a mechanism involving Mu-mediated replicon fusion. Also, heterospecific matings between L. pneumophila Trp+ and Thy+ exconjugants and trp and thy auxotrophs of E. coli showed that complementation by R prime plasmids was not responsible for the Trp+ and Thy+ phenotypes of the exconjugants. One disadvantage of using Mu-containing plasmids for chromosome mobilization is that transfer of chromosomal markers usually does not occur in a polar, oriented fashion from a single fixed origin of transfer. Instead, chromosome Microbes and Infection 1999, 1203-1209
transfer usually proceeds from multiple origins in different donors, which makes co-transfer and linkage experiments difficult to interpret. To overcome this potential problem, we sought to use transposon-facilitated recombination (Tfr) to mobilize the L. pneumophila chromosome. With Tfr [27], regions of DNA sequence homology are created between a conjugative plasmid and the donor chromosome by introducing identical transposable elements into each of the replicons (figure 1). Integration of the plasmid via homologous recombination at the site of the insertion in the donor chromosome is thought to promote an oriented transfer of chromosomal markers using the integration site as the sole origin of chromosome transfer. In a previous study [19], we introduced more than 25 independent Mu insertions into the Philadelphia 1 chromosome using the mini-Mu fusion phage MudI1681. Southern hybridization experiments confirmed that each of the insertions mapped to a different region of the Philadelphia 1 chromosome. Further, several strains that contained MudI1681 chromosomal insertions also harbored the plasmid that was originally used to introduce MudI1681 into strain Philadelphia 1. This provided us with an ideal opportunity to determine whether Tfr could be used to promote oriented transfer of chromosomal markers in L. pneumophila. To test this, we performed mating experiments with several of these MudI1681containing strains as donors and a double auxotrophic mutant (trp and thy) as a recipient [23]. The results of these experiments showed that Tfr donors transferred the trp locus at a higher frequency than donors that harbored Mu-containing plasmids alone ([23], table II). Transfer of the thy locus varied between donors [23]. Unfortunately, we never observed co-transfer of the Thy+ and Trp+ markers during Tfr mating experiments. The lack of linkage between the thy and trp loci indicated that it would be necessary to create additional double and triple auxotrophic strains to determine whether Tfr could mediate the oriented transfer of chromosomal markers in L. pneumophila. Nevertheless, our results indicated that Tfr can be used to promote transfer of chromosomal genes in L. pneumophila and that the thy and trp loci were not in close proximity to one another on the L. pneumophila chromosome. In the early 1980s, Simon [33] constructed the Tn5Mob transposon that contains the Mob region of the IncP plasmid RP4. He reported that Gram-negative bacteria that contained Tn5-Mob chromosomal insertions and a helper IncP plasmid (to supply necessary transfer proteins in trans) could transfer chromosomal markers in a polar, oriented fashion during conjugation (figure 2). The Tn5Mob system offered an advantage over Mu-based Tfr because, unlike Mu, Tn5 is not capable of secondary transposition in L. pneumophila. The inability of Tn5-Mob to undergo secondary transposition in L. pneumophila insured that chromosomal gene transfer in Tn5-Mobcontaining donors occurred from a single fixed origin of transfer (rather than from multiple origins) during conjugation. To test this, we introduced several Tn5-Mob insertions in strain Philadelphia 1 that mapped to different regions of the L. pneumophila chromosome [23]. Following intro1205
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Figure 1. Schematic representation of transposon-facilitated recombination 1. Regions of homology are created in the donor by Mu insertions found in the self-transmissible IncP plasmid and L. pneumophila chromosome. 2. Homologous recombination between plasmid and chromosomal Mu insertions. 3. IncP plasmid is integrated into the L. pneumophila chromosome during recombination. 4. The origin of plasmid transfer (oriT) of the integrated plasmid serves as the origin of chromosome transfer which results in the transfer of chromosomal genes from donor to recipients during conjugation.
duction of a helper IncP plasmid, mating experiments were performed between Tn5-Mob donors and several auxotrophic recipients, e.g., guanine (gua), trp, and thy,. Although Tn5-Mob donors transferred each of the abovementioned chromosomal markers, recombination frequencies in recipients were 10-fold lower than those obtained with the Mu-based Tfr system [23]. The reason for differences in the recombination frequencies of selected markers between the Mu-based Tfr and Tn5-Mob systems is not clear. It is possible that the direction of chromosome transfer or location of Tn5-Mob insertions relative to the selected markers could account for the observed differences. Nonetheless, our results clearly demonstrated that three different chromosome mobilization systems could be employed to develop a conjugationbased system of gene transfer for L. pneumophila.
4. Identification of indigenous conjugative plasmids As mentioned above, a number of indigenous plasmids ranging in size from 35 to 128 kb have been identified in L. pneumophila (table III). Many of these plasmids were originally identified in serogroup 1 strains [10, 14, 18]. This led several investigators to speculate that virulence 1206
genes may be carried on the plasmids, because the majority of L. pneumophila isolates from patients with Legionnaires’ disease were from serogroup 1. However, since most serogroup 1 clinical isolates did not contain plasmids, it was generally assumed that plasmids were not required for virulence. Consequently, these plasmids were not studied in much detail following their identification. Several groups had reported that a number of environmental and clinical serogroup 1 strains harbored a 128-kb plasmid [10, 14]. This observation, along with our previous work which showed that conjugation was possible in L. pneumophila, led us to investigate the conjugative properties of the 128-kb plasmid, which we called pCH1. Mating experiments between legionellae that harbored pCH1 and plasmidless recipients revealed that pCH1 was self-transmissible by conjugation to several serogroup 1 strains of L. pneumophila. Plasmid transfer frequencies ranged from 10–3 to 10–4 per recipient [22]. Interestingly, pCH1 could not be transferred by conjugation from serogroup 1 donors to serogroup 3 strains or E. coli. Additional mating experiments between legionellae that harbored pCH1 and several auxotrophic recipients (serogroups 1 and 3) showed that pCH1 did not promote transfer of three chromosomal markers (gua, thy or trp). Despite numerous attempts, we were unable to identify a phenotype conferred upon strains that harbored pCH1. Microbes and Infection 1999, 1203-1209
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Figure 2. Tn5-Mob mediated mobilization of the L. pneumophila chromosome. 1. Tn5-Mob transposon is introduced into L. pneumophila on a suicide vector. 2. Tn5-Mob transposes from the suicide vector into the L. pneumophila chromosome. 3. A helper IncP plasmid is transferred into the donor that contains a Tn5-Mob chromosomal insertion. The helper IncP plasmid supplies all of the necessary transfer functions (in trans) that are required for chromosome mobilization and gene transfer. 4. Mob sequences contained in Tn5-Mob transposon act as the origin of chromosome transfer to transfer chromosomal markers during conjugation.
In a similar study, Tully [35] showed that a 55-kb plasmid found in a clinical serogroup 1 strain called Dodge was self transmissible by conjugation to other serogroup 1 isolates. Like pCH1, this plasmid could not be transferred by conjugation from L. pneumophila to E. coli or P. aeruginosa. The plasmid was compatible with plasmids of the IncP and IncW incompatibility groups. Further, strains that harbored the plasmid exhibited increased resistance to killing by ultraviolet light as compared with isogenic plasmidless isolates. Another serogroup 1 plasmid, designated pLPG36, was studied in great detail by Lopez de Felipe [13]. He demonstrated that one of four recombinant plasmids
constructed from pLPG36 in pUC18 could be mobilized by IncP plasmids. Unfortunately, Lopez de Felipe failed to determine whether the native plasmid, pLPG36, was self transmissible by conjugation. Again, as was the case with pCH1, there was no obvious phenotype associated with pLPG36. Nevertheless, the results from our work and that of Tully indicated that serogroup 1 strains of L. pneumophila harbor indigenous plasmids that are self transmissible by conjugation. Furthermore, it is possible that integration of indigenous plasmids into the L. pneumophila chromosome may promote the transfer of chromosomal genes during conjugation.
Table III. Indigenous plasmids found in L. pneumophila. Designation pCH1 pLP3 pUH2 pLPG36 Dodge pUPH1 pLp4269
Size (kb)
Conjugative
Phenotype
Sourcea
Reference
128 92 69 58 55 50 37.5
+ ND ND ND + ND ND
cryptic cryptic ND cryptic UV resistance ND ND
E/C C E E/C C E/C E/C
[22] [18] [14] [13] [35] [15] [14]
a
Bacteria that harbored plasmids were isolated from either clinical or environmental samples. C, clinical; E, environmental; ND, not determined.
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5. Conjugation and intracellular growth The identification of genes that permit L. pneumophila to survive and grow intracellularly within eukaryotic host cells has been a subject of intense research over the past five years. To date, two groups (Howard Shuman’s at Columbia University and Ralph Isberg’s at Tufts University), have identified 23 genes located in two unlinked regions of the L. pneumophila chromosome that are necessary for the intracellular growth of this organism. The Shuman group calls these genes icm (for ‘intracellular multiplication’), whereas the Isberg group calls them dot (for ‘defect in organelle trafficking’). All 23 genes have been cloned and their DNA sequences determined [16, 29, 30–32]. Sequence analysis of proteins encoded by icm genes revealed that four of the Icm proteins (IcmP, IcmO, IcmL and IcmE) contained substantial amino acid similarity to plasmid-encoded genes involved in DNA transfer [30–32, 36]. An additional protein (DotB) was found to be homologous to a large family of nucleotide-binding proteins that include members of various conjugal transfer systems [36]. This suggested that an endogenous conjugation system may exist in L. pneumophila and that wild-type strains of L. pneumophila may be able to mediate plasmid DNA transfer. To test this, the Shuman and Isberg groups introduced derivatives of the non-self-transmissible plasmid IncQ plasmid RSF1010 into several wild-type donors and performed mating experiments with appropriately marked antibiotic-resistant recipients. Both groups found that RSF1010 could be easily transferred via conjugation [32, 36]. Further, they showed that conjugation was dependent on certain icm/dot genes, because mutations in these genes interfered with conjugation [32]. Of interest, in a recent report, Segal and Shuman [31] showed that a functional RSF1010 mobilization system in wild-type strains of L. pneumophila was required for intracellular growth in macrophage-like cells. Both groups [31, 36] have proposed that the icm/dot conjugation system plays a key role in the intracellular growth of L. pneumophila by transporting an effector molecule(s) into host cells that inhibits phagosome-lysosome fusion. This, in turn, allows L. pneumophila to multiply unabated within unfused phagosomes. The natural substrate for icm/dot system is likely to be a protein, rather than DNA, because inhibition of phagosome-lysosome fusion occurs within 30 minutes following ingestion of legionellae and there is probably not enough time for DNA to be transferred and expressed within this time period [31].
6. Concluding remarks It is evident from this review that our understanding of the mechanisms of gene transfer in L. pneumophila has increased dramatically in recent years. For example, we now know that L. pneumophila 1) is capable of natural DNA transformation and can be transformed by electroporation, 2) harbors indigenous conjugative plasmids and 3) possesses a chromosome-based, endogenous system of 1208
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plasmid transfer. Nevertheless, additional research will be required to determine whether L. pneumophila utilizes one or more of these mechanisms to promote gene transfer in nature. The advent of bacterial genome sequencing has rendered conventional methods of gene mapping obsolete. Therefore, it is unlikely that transformation or conjugation will be considered when constructing a genetic and physical map of the L. pneumophila chromosome. Nevertheless, identification of the icm/dot system of plasmid transfer raises some important fundamental questions regarding gene transfer in L. pneumophila. First, can plasmids other than those belonging to the IncQ incompatibility group be mobilized by the icm/dot system? Second, are indigenous conjugative plasmids dependent on icm/dot or truly self transmissible by conjugation? Finally can the icm/dot system promote the transfer of chromosomal genes in L. pneumophila? Answers to these questions will undoubtedly enhance our understanding of the genetic organization and underlying pathogenic mechanisms of this intriguing intracellular pathogen.
Acknowledgments I want to thank Chang Hua Zou for excellent technical assistance and her dedication to the study of gene transfer in L. pneumophila. The artistic talents of Vincent Racaniello and the helpful and insightful discussions with Dave Figurski are greatly appreciated and acknowledged. Finally, I want to thank Howard Shuman for teaching me everything I ever wanted to know about bacterial genetics and then some.
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[23] Mintz C.S., Zou C.H., Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob, Can. J. Micro. 38 (1992) 664–671. [24] Newsome A.L., Baker R.L., Miller R.D., Arnold R.R., Interactions between Naeglaria fowleri and Legionella pneumophila, Infect. Immun. 50 (1985) 449–452. [25] Nolte F.S., Conlin C.A., Roisin A.J., Redmond S.R., Plasmids as epidemiological markers in nosocomial Legionnaires’ disease, J. Infect. Dis. 149 (1984) 251–256. [26] Pearlman D., Jiwa A.H., Engleberg N.C., Eisenstein B.I., Growth of Legionella pneumophila in a human macrophagelike (U937) cell line, Microb. Pathogen. 5 (1988) 87–95. [27] Pischl D.L., Farrand S.K., Transposon-facilitated chromosome mobilization in Agrobacterium tumemfaciens, J. Bacteriol. 153 (1983) 1451–1460. [28] Rowbotham T.J., Current views on the relationship between amoebae, legionellae and man, Israel. J. of Med. Sci. 22 (1986) 678–689. [29] Roy C.R., Berger K.H., Isberg R.R., Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur with minutes of bacterial uptake, Mol. Micro. 28 (1998) 663–674. [30] Segal G., Shuman H.A., Characterization of a new region required for macrophage killing by Legionella pneumophila, Infect. Immun. 65 (1997) 5057–5066. [31] Segal G., Shuman H.A., Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of the IncQ plasmid RSF1010, Mol. Micro. 30 (1998) 197–208. [32] Segal G., Purcell M., Shuman H.A., Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome, Proc. Natl. Acad. Sci. USA 95 (1998) 1669–1674. [33] Simon R., High frequency mobilization of gram negative replicons by the in vitro constructed Tn5-Mob transposon, Mol. Gen. Genet. 196 (1994) 413–420. [34] Stone B.J., Abu Kwiak Y., Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili, J. Bacteriol. 181 (1999) 1395–1402. [35] Tully M., A plasmid from a virulent strain of Legionella pneumophila is conjugative and confers resistance to ultraviolet light, FEMS Microb. Lett. 90 (1991) 43–48. [36] Vogel J.P., Andrews H.L., Wong S.K., Isberg R.R., Conjugative transfer by the virulence system of Legionella pneumophila, Science 279 (1998) 873–876. [37] Wiater L.A., Marra A., Shuman H.A., Escherichia coli F plasmid transfers to and replicates within Legionella pneumophila: an alternative to using an RP4-based system for gene delivery, Plasmid 32 (1994) 280–294.
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