Abstract
In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 × 109 CFU) and Chicago-2S (approximately 8.9 × 109 CFU) were mated typically yielded 103 Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.
Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular bacterium that can grow in human monocytes (8) and alveolar macrophages (17) and in the macrophages of guinea pigs (10). Two unlinked 20-kb regions of genes required for human macrophage killing and intracellular multiplication have been identified on the L. pneumophila chromosome; they have been designated icm (intracellular multiplication) (11, 21) and dot (defect in organelle trafficking) (2). Region I contains seven genes (icmV, -W, and -X and dotA, -B, -C, and -D) (1, 5, 27, 28), and region II contains 16 genes (icmT, -S, -R, -Q, -P, -O, -N, -M, -L, -E, -G, -C, -D, -J, -B, and-F) (1, 18, 22, 23, 24). All of these genes have also been shown to be required for intracellular growth in Acanthamoeba castellanii (25).
We previously reported on chromosomal conjugation as a novel DNA transfer system in L. pneumophila (14). In brief, virulent strain K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1 and belongs to serogroup 1, was used as one parent, and avirulent strain Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 and belongs to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 × 109 CFU/ml) and Chicago-2S (approximately 8.9 × 109 CFU/ml) were mated typically yielded 103 CFU of Kmr Smr LacZ+ recombinants per ml, corresponding to a transfer frequency of 10−6 per parent. All of the recombinants tested (100 of 100) belonged to serogroup 6, which is the same serogroup as strain Chicago-2S. Genotyping of both parents and their recombinants by repetitive-element PCR, arbitrarily primed PCR, and pulsed-field gel electrophoresis (PFGE) revealed an asymmetric role for each parent; that is, strain K6 is a donor, and strain Chicago-2S is a recipient. In addition, Southern hybridization analysis made clear that chromosomal genes transferred from K6 were integrated into the chromosome of Chicago-2S by homologous recombination. We speculated that some of the genes responsible for the growth of strain K6 within amoebae or macrophages might be transferred to strain Chicago-2S and that they might be integrated into the Chicago-2S chromosome by recombination. If this were so, some transconjugants would be able to grow within amoebae or macrophages. In this study, we examined whether virulence conversion occurs in L. pneumophila by conjugal transfer of chromosomal DNA.
The L. pneumophila strains and plasmids used in this study are listed in Table 1. L. pneumophila was grown in BYE broth (19) and on BCYE agar plates (6). Antibiotics for L. pneumophila selection were used at the following concentrations: kanamycin, 50 μg/ml; streptomycin, 50 μg/ml; rifampin, 100 μg/ml; chloramphenicol, 5 μg/ml; gentamicin, 50 μg/ml. L. pneumophila strain Chicago-2 or its derivative, Chicago-2S, was avirulent, although strain Chicago-2 was first isolated from a patient's lungs (12). Chicago-2 has lacked the ability to grow intracellularly in macrophages, but we do not know why this is so. There was no large deletion in the icm-dot genes of strain Chicago-2S (unpublished observation), and there was no severe defect in its type II secretion system (unpublished observation). Even when it was inoculated (8.4 × 108 CFU) into guinea pigs intraperitoneally, the bacterium could not be isolated from the guinea pig spleens on day 3 or 4 after infection. Growth and maintenance of A. castellanii ATCC 30324 in Proteose Peptone-yeast extract-glucose medium (4, 15) in 75-cm2 tissue culture flasks were performed as previously described (4, 15).
TABLE 1.
L. pneumophila strains and plasmids used in this study
| Strain or plasmid | Genotype or phenotype | Reference or source |
|---|---|---|
| Strains | ||
| 25D | Avirulent mutant of Philadelphia-1; Icm− | 9 |
| AM511 | Philadelphia-1 Smr r− m+ | 12 |
| Chicago-2 | Avirulent, serogroup 6; Icm− | ATCC 33215 |
| Chicago-2S | Streptomycin-resistant derivative of Chicago-2 | 14 |
| Chicago-2SR | Rifampin-resistant derivative of Chicago-2S | This study |
| GS3001 | JR32 icmS3001::Kmr (icm-dot region II) | 22 |
| GS3008 | JR32 icmM3008::Kmr (icm-dot region II) | 24 |
| GS3010 | JR32 icmK3010::Kmr (icm-dot region II) | 24 |
| HM1011-HM1013 | Recombinants between K6 and Chicago-2S; Icm+ serogroup 6 | 14 |
| HM1014 | Recombinant between K6 and Chicago-2S; Icm− serogroup 6 | 14 |
| HM1042-HM1044 | Recombinants between K6 and Chicago-2S; Icm− serogroup 6 | 14 |
| HM1134 | LELA3473 with pGS-Lc32 | This study |
| HM1138 | LELA3393 with pMW100 | This study |
| HM1156-HM1164 | Recombinants between LELA2883 and Chicago-2SR; Icm− serogroup 6 | This study |
| HM1175 | Chicago-2SR with pMMB207C | This study |
| JR32 | Salt-sensitive isolate of AM511 | 21 |
| K6 | Philadelphia-1 chromosome::Tn903dIIlacZ | 14 |
| LELA2883 | JR32 dot B2883::Tn903dIIlacZ (icm-dot region I) | 21 |
| LELA2883-28 | LELA2883 with Gm insertion instead of lvh region | 26 |
| LELA3118 | JR32 dotA3118::Tn903dIIlacZ (icm-dot region I) | 21 |
| LELA1747 | JR32 icmV1747::Tn903dIIlacZ (icm-dot region I) | 21 |
| LELA4004 | JR32 icmX4004::Tn903dIIlacZ (icm-dot region I) | 21 |
| LELA3473 | JR32 icmR3473::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA3463 | JR32 icmO3463::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA3352 | JR32 icmP3352::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA4032 | JR32 icmO4032::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA4432 | JR32 icmE4432::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA4432-28 | LELA4432 with Gm insertion instead of lvh region | 26 |
| LELA3244 | JR32 icmD3244::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA3393 | JR32 icmB3393::Tn903dIIlacZ (icm-dot region II) | 21 |
| LELA1718 | JR32 icmF1718::Tn903dIIlacZ (icm-dot region II) | 21 |
| MW635 | JR32 icmG635::Kmr (icm-dot region II) | 18 |
| MW656 | JR32 icmJ656::Kmr (icm-dot region II) | 18 |
| Philadelphia-1 | Virulent, serogroup 1 | ATCC 33152 |
| Plasmids | ||
| pGS-Lc-32 | icmTSRQPO in pMMB207 | 22 |
| pGS-Lc-47 | icmNMLKEG in pMMB207αb | 24 |
| pLAW317 | rpsL MCSaoriT(RK2) CmrloxP oriR(ColE1) AprloxP | 30 |
| pLAW330 | pLAW317::Tn903dIIlacZ tnpA(Tn903) oriR(F1) | 30 |
| pMMB207 | RSF1010 derivative; IncQ lacIq Cmr Ptac oriT | 16 |
| pMMB207αb | pMMB207 containing MCS (α complementation) | 22 |
| pMMB207C | pMMB207 with 8-bp insertion in mobA; Mob− | G. Segal and H. A. Shuman, unpublished data |
| pMW100 | icmGCDJBF and tphA in pMMB207 | 18 |
MCS, multiple cloning site.
Intracellular growth of transconjugants in A. castellanii.
The ability of transconjugants to grow within A. castellanii was screened by a spot assay in which 106 amoebae were spread on a BCYE plate for screening of the intracellular growth of transconjugants in amoebae. Individual colonies of L. pneumophila were spotted with a toothpick onto a BCYE plate with nothing added and onto a second BCYE plate on which amoebae had been spread. The plates were incubated at 28°C for 4 to 5 days and then visually inspected for the growth of each spot of L. pneumophila. Strains Philadelphia-1 and JR32 grew equally well on both plates. Strains Chicago-2 and 25D, which are unable to grow within A. castellanii, did not form visible growth on the BCYE plate spread with amoebae. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6, that is, the same serogroup as Chicago-2S, formed visible growth on the BCYE plate spread with amoebae. Serogroups of transconjugants were determined by slide agglutination tests with monoclonal (Monoclonal Technologies, Inc., Atlanta, Ga.) or polyclonal (Denka Seiken Co., Ltd., Tokyo, Japan) antibodies against serogroups 1 and 6. All of these 32 strains belonged to serogroup 6. On the basis of these results, to evaluate the intracellular growth in the amoebae quantitatively, seven transconjugants originating from three separate conjugation experiments were selected from the 1,152 transconjugants and used for experiments. Strains HM1011, HM1012, and HM1013 formed visible colonies on amoeba-containing agars as well as strain K6 did, and the other strains (HM1014, HM1042, HM1043, and HM1044) did not grow on the agar plates as well as Chicago-2S did. Intracellular growth assays were performed as previously described (15), with some modifications. L. pneumophila was added at a multiplicity of infection of 10 to an adherent monolayer of 1.5 × 105 amoebae. After incubation for 30 min at 37°C to allow for infection, the wells were washed three times with 0.5 ml of Ac buffer (15). A sample of the infection supernatant was removed once every 24 h for 4 days. Numbers of CFU of extracellular bacteria on BCYE plates were quantified. As the Ac buffer does not support the growth of L. pneumophila, the CFU represent bacteria that have grown within the amoebae. As shown in Fig. 1A, strain HM1011 grew in the amoebae approximately 105-fold in 2 days, i.e., as well as K6 did, while strain HM1014 did not grow in the amoebae as well as Chicago-2S did. Although the data are not included here, we observed that strains HM1012 and HM1013 grew in the amoebae as well as strain K6 did, while strains HM1042, HM1043, and HM1044 did not grow in the amoebae as well as strain Chicago-2S did.
FIG. 1.
Time course of intracellular growth of L. pneumophila strains in A. castellanii (A) and in peritoneal macrophages of guinea pigs (B). The experiments were done at least three times, and the data are means ± standard errors. Symbols: •, K6; ○, Chicago-2S; ▵, HM1011; □, HM1014.
Intracellular growth of transconjugants within guinea pig macrophages.
It was confirmed by light microscopic observation of Gimenez-stained macrophages infected with the 32 transconjugants that all of the transconjugants could grow in guinea pig macrophages as well. Quantitative assay of intracellular growth in guinea pig macrophages (Fig. 1B) was performed with the same strains as described in Fig. 1A. The intracellular growth of L. pneumophila in peritoneal macrophages from guinea pigs was examined after in vitro phagocytosis. Peritoneal exudate cells were collected by lavage of the peritoneal cavities of guinea pigs 4 days after intraperitoneal injection of 10 ml of 10% Proteose Peptone (Difco). The preparation of peritoneal macrophage monolayers from the peritoneal exudate cells, in vitro phagocytosis, and the microscopic observation of Gimenez-stained macrophage monolayers were carried out as described previously (13). L. pneumophila was added at a multiplicity of infection of 10 to monolayers of 106 macrophages. After incubation for 1.5 h at 37°C to allow for in vitro phagocytosis, the wells were washed three times with sterile phosphate-buffered saline (PBS; 136.8 mM NaCl, 2.7 mM KCl, 8.1 mM NaH3PO4, 1.5 mM KH3PO4) to remove nonphagocytosed bacteria. The infected macrophages were incubated at 37°C in a CO2 incubator, and bacterial CFU were determined 0, 24, 48, and 72 h after infection. The number of CFU in a whole well was determined by removing the culture medium, adding 0.1 ml of sterile water to the cell monolayer, combining the two fractions, and plating samples on BCYE plates. Strain HM1011 grew approximately 103-fold in the macrophages in 2 days, i.e., as well as strain K6 did, while strain HM1014 did not grow in the macrophages as well as Chicago-2S did (Fig. 1B). Although the data are not included here, strains HM1012 and HM1013 grew in the macrophages as well as strain K6 did, while strains HM1042, HM1043, and HM1044 did not grow in the cells as well as strain Chicago-2S did. These results show that some of the genes on the strain K6 chromosome that are responsible for its growth within amoebae and macrophages are transferred to Chicago-2S, and they are integrated into the Chicago-2S chromosome. These results strongly suggest that avirulent strain Chicago-2S may change into a virulent strain.
Infection of guinea pigs by Legionella aerosols.
To assess the virulence of L. pneumophila strains, guinea pigs were infected with sublethal doses (approximately 105 CFU) of Legionella aerosols generated from a suspension of strain K6, Chicago-2S, or HM1011. Female guinea pigs of the outbred Hartley strain, weighing 250 to 350 g, were purchased from Shizuoka Experimental Animals (Hamamatsu, Japan). The protocols of the animal experiments were approved by the institutional animal care committee of the University of Occupational and Environmental Health. L. pneumophila strains were harvested from BCYE plates after 48 h of growth and suspended in BYE medium. The cultures were grown at 37°C in a shaking incubator to late log phase. The bacteria were recovered by centrifugation, washed twice with sterile PBS, and then suspended at approximately 5 × 108 CFU/ml in sterile PBS, which corresponds to a sublethal dose. Legionella aerosols were generated from the bacterial suspensions by an ultrasonic nebulizer (NE-U12; OMRON Co., Ltd., Tokyo, Japan). We confirmed that about 70% of the aerosols generated by the nebulizer resulted in less than 6 μm reaching alveoli and that ultrasonication by the nebulizer for 30 min did not affect the numbers of CFU in the bacterial suspension set in the machine (data not shown). The aerosols generated were transported at 7 liters/min for 15 min by a vacuum pump into an all-glass metabolic chamber (volume, 7 liters; Shibata Scientific Technology, Ltd., Tokyo, Japan) into which animals were put. The vacuum speed (7 liters of air per min) was chosen to keep constant concentrations of Legionella aerosols in the chamber during exposure. After exposure to Legionella aerosols, the body weight and rectal temperature of each animal were measured daily. The left lungs were harvested aseptically on days 0 (2 h after infection), 2, 4, and 7 postinfection, and their homogenates were cultured quantitatively on BCYE agar. The right lungs were fixed in 10% formalin for more than 2 weeks before dissection. Three or four of these sections were stained with hematoxylin and eosin and examined microscopically. Animals infected with strain K6 or HM1011 became febrile 2 days after infection. Their body temperatures rose to a maximum at 4 or 5 days after infection and then fell to normalcy by day 8. Animals infected with strain Chicago-2S did not become febrile. Animals infected with K6 or HM1011 had an average weight loss of 15% by day 5 after infection, in contrast to the 7% weight gain of animals infected with strain Chicago-2. Approximately 5 × 104 CFU of bacteria were recovered from the left lungs of animals infected with strain K6 or HM1011 at 2 h after infection. Numbers of CFU in the left lungs increased about 104-fold in 4 days after infection, reached a maximum of approximately 9 × 108 CFU, and decreased thereafter. Approximately 2 × 104 CFU of bacteria were recovered from the left lungs of animals infected with strain Chicago-2S at 2 h after infection, but the bacteria were not detected (<102 CFU) 2 days after infection. Histological examination of their right lungs revealed a greater degree of exudation in animals infected with strain K6 (Fig. 2A) or HM1011 (Fig. 2C) than in those infected with strain Chicago-2S (Fig. 2B) on day 4 or 7 postinfection. These results show that strain Chicago-2S changed to virulent strain HM1011.
FIG. 2.
Light micrographs of hematoxylin-and-eosin staining of the right lungs of guinea pigs sublethally infected with Legionella aerosols. The right lungs were harvested at 4 days after infection. After fixing and staining, light microscopic observations were performed (magnification, ×40). Panels: A, strain K6; B, strain Chicago-2S; C, strain HM1011. In contrast to those in panel B, alveolar spaces in panels A and C are occupied by many inflammatory cells (arrows).
Conjugal transfer of chromosomal virulence genes (icm-dot genes).
To learn about transfer of chromosomal virulence genes more precisely, we examined whether the icm-dot genes can be transferred. Mating experiments were performed with donor strains with the genes for Kmr or Kmr LacZ+ at different sites within the icm-dot genes of the strain JR32 chromosome (Table 1). As a recipient strain, a spontaneous Rifr derivative of strain Chicago-2S (strain Chicago-2SR) was used because donor strains were Smr. Mating experiments were performed as previously described (14). Selection for recombinants was performed on agar plates containing both kanamycin and rifampin, and top agar containing 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside was also used when LELA strains were used as donors in the experiments. Table 2 shows the results obtained with LELA2883 (dotB) as the donor. The transfer frequency corresponded to about 10−4 per donor (Table 2, line 1). No Kmr Rifr LacZ+ colonies were obtained when only Chicago-2SR was plated (Table 2, line 7), and spontaneous Rifr mutants were obtained when LELA2888 was plated on selective media (Table 2, line 6). No recombinants were obtained when the mating experiments were carried out with BYE broth (Table 2, line 3), implying that extended cell-to-cell contact or a high cell density is required for efficient transfer. Recombinant formation was not affected by DNase I (10 μg/ml) (Table 2, line 2), and no transfer was seen with one viable parent and one heat-killed parent before mating (Table 2, lines 3 and 4). No recombinants were obtained by mixing and incubating cell-free filtrates of one parent with the cells of the other parents, and no plaques were detected if filtrate from one parent was spotted onto a lawn of the other parent. These results suggested that transformation or transduction was not involved in the recombinant formation observed. The mechanism of the DNA transfer seen was most consistent with conjugal transfer.
TABLE 2.
Transfer of chromosomal antibiotic resistance markers from L. pneumophila strain LELA2883 to Chicago-2SRa
| Serogroup 1, Kmr LacZ+ donor | Serogroup 6, Rifr recipient | Mating condition(s) | No. of Kmr Rifr LacZ+ recombinants/ml | Transfer frequency (no. of recombinants/donor) |
|---|---|---|---|---|
| LELA2883 | Chicago-2SR | BCYE agar | (5.1 ± 2.3) × 105 | (1.7 ± 0.4) × 10−4 |
| LELA2883 | Chicago-2SR | BCYE agar, DNase I | (2.7 ± 0.6) × 105 | (2.2 ± 1.5) × 10−4 |
| LELA2883 | Chicago-2SR | BYE broth | 0 | |
| LELA2883 | Chicago-2SR | BCYE agar, heat-killed LELA2883 | 0 | |
| LELA2883 | Chicago-2SR | BCYE agar, heat-killed Chicago-2SR | (2.0 ± 1.7) × 102b | (1.4 ± 0.7) × 10−7c |
| LELA2883 | BCYE agar | (2.1 ± 1.0) × 102b | (1.3 ± 1.4) × 10−7c | |
| Chicago-2SR | BCYE agar | 0 |
The numbers of recombinants and transfer frequencies are based on at least three experiments and were calculated as means ± standard deviations. The numbers of LELA2883 and Chicago-2SR CFU were (2.6 ± 1.1) × 109/ml and (5.0 ± 1.8) × 109/ml at the end of the experiments, respectively.
Number of spontaneous mutations of LELA2883 to Rifr.
Frequency of spontaneous mutation of LELA2883 to Rifr.
To genetically confirm the DNA transferred from LELA2883 to Chicago-2SR, genotyping of strains LELA2883 and Chicago-2SR and their transconjugants was performed by PFGE as described previously (14). As shown in Fig. 3A, all of the transconjugants (HM1156 to HM1164) exhibited identical restriction fragment length polymorphism after PFGE of SfiI-digested genomic DNA. The restriction fragment patterns of the transconjugants were distinct from the pattern exhibited by LELA2883 and were similar to that exhibited by Chicago-2SR (Fig. 3A, lanes 2 to 11). These findings show that strain LELA2883 is the donor and strain Chicago-2SR is the recipient and also suggest that chromosomal genes transferred from LELA2883 are integrated into the Chicago-2SR chromosome by recombination. To examine the presence of the LELA2883 sequences in the Chicago-2SR chromosome, we performed Southern blot experiments with pLAW330 (30) as a probe. Prehybridization, hybridization (at 68°C overnight), and chemiluminescent detection of the nylon membrane (Hybond-N+; Amersham Japan Co., Ltd., Tokyo, Japan) blots were performed with a DIG DNA labeling and detection kit (Boehringer GmbH, Mannheim, Germany) in accordance with the instructions of the manufacturer. As shown in Fig. 3B, the genes for Kmr and LacZ+ were located on an about 582-kb SfiI-digested DNA fragment of strain LELA2883 (Fig. 3B, lane 1). In all of the transconjugants tested (HM1156 to HM1164), the genes were located on an about 582-kb SfiI-digested DNA fragment (Fig. 3B, lanes 3 to 11). This is direct evidence that chromosomal genes transferred from LELA2883 are integrated into the chromosome of Chicago-2SR. Some bands of more than 582 kb are probably due to partial digestions, as well as bands observed at their sample plugs. In addition, divergences in signal intensity between these samples are due to differences in the amount of DNA electrophoresed in the gel.
FIG. 3.
Cleavage patterns (A) and Southern blot analysis (B) of SfiI-digested genomic DNAs of L. pneumophila strains by PFGE. Plasmid pLAW330 digested with HindIII, which does not cut within Tn903dIIlacZ, was labeled with digoxigenin and used as a probe. Lanes: M, lambda ladder (Bio-Rad), used as a DNA size standard; 1, LELA2883 (dotB); 2, Chicago-2SR; 3, HM1156; 4, HM1157; 5, HM1158; 6, HM11159; 7, HM1160; 8, HM1161; 9, HM1162; 10, HM1163; 11, HM1164.
Figure 4 shows the results obtained in these conjugation experiments. Recombinant formations were not affected by DNase I (10 μg/ml). Frequencies (mean ± standard deviation) of spontaneous mutation to Rifr for donor strains of icmX and -V; dotA and -B; icmF, -B, -E, -P, and -R; and icmS were (4.9 ± 3.9) × 10−7 and (1.0 ± 0.8) × 10−7; (0.8 ± 1.4) × 10−7 and (1.3 ± 1.4) × 10−7; (1.7 ± 0.6) × 10−7, (3.2 ± 4.1) × 10−7, (4.5 ± 2.3) × 10−7, (3.4 ± 2.6) × 10−7, and (3.0 ± 3.2) × 10−7; and (1.3 ± 1.2) × 10−7, respectively. Although the data are not included in Fig. 4, strains with insertions in seven icm genes (icmQ, -O, -M, -K, -G, -D, and -J) had transfer frequencies of 10−7 to 10−6. When it was found that the spontaneous mutation of donor strains to Rifr was not far off the conjugal frequency, it was confirmed that all (50 of 50) of the transconjugants tested belonged to serogroup 6, which is the same serogroup as Chicago-2SR. To distinguish transconjugants from Rifr mutants of donor strains more clearly, we introduced a nonconjugative Cmr plasmid (pMMB207C) as a cytoplasmic marker into Chicago-2SR. Kmr Rifr (LacZ+) recombinants were selected after mating of Chicago-2SR containing pMMB207C (HM1175, Rifr Cmr) with Kmr (LacZ+) donor strains. Transconjugants were screened for the presence of the Cmr plasmid by patching onto plates containing chloramphenicol. All of the Kmr Rifr (LacZ+) recombinants tested (100 of 100) were Cmr and belonged to serogroup 6. Their conjugal frequencies were similar to the results shown in Fig. 4 (data not shown). These results suggest that all of the genes for Kmr or Kmr LacZ+ within the icm-dot genes were horizontally transferred into strain Chicago-2SR, indicating that the icm-dot locus can be transferred by the chromosomal conjugation system. Differences in transfer efficiency between icm-dot genes observed in Fig. 4 may reflect distances from chromosomal oriT to the insertion sites of the genes for Kmr (LacZ+) in the strain LELA, GS, and MW chromosomes. The chromosomal oriT locus may be closer to the region around dotB-dotA than to the rest of the icm genes.
FIG. 4.
Frequencies of conjugal transfer of the icm-dot locus. Chicago-2SR was used as a recipient in all of the conjugation experiments. The donor strains tested were LELA4004 for icmX, LELA1747 for icmV, LELA3118 for dotA, LELA2883 for dotB, LELA1718 for icmF, LELA3393 for icmB, HM1138 for icmB/pMW100, LELA4432 for icmE, LELA3352 for icmP, LELA3473 for icmR, HM1134 for icmR/pGS-Lc32, GS3001 for icmS, LELA2883-28 for dotB lvh, and LELA4432-28 for icmE lvh. The other strains tested containing insertions in icmQ, -O, -M, -K, -G, -D, and icmJ are listed in Table 1. The experiments were done at least three times. The values are means ± standard deviations.
In addition, this result suggests that the icm-dot apparatus may not be involved in the chromosomal DNA transfer observed because all of the donor strains used here have defects in the icm-dot apparatus. When plasmid pGS-LC-32 was introduced into strains GS3001 (icmS), LELA3473 (icmR), and LELA3463 (icmQ), this plasmid complemented them for intracellular growth in guinea pig macrophages (data not shown), indicating that the icmS, icmR, or icmQ gene product is expressed from this plasmid and the intact icm-dot apparatus is reconstructed. However, no change in the transfer frequency of the chromosomal DNA was observed when these complemented strains were used as donors for the conjugation experiments (icmR and icmR/pGS-Lc32 in Fig. 4). When plasmid pGS-Lc-47 or pMW100 was introduced into LELA4432 (icmE) or LELA3393 (icmB), respectively, the transfer frequencies of the complemented strains were the same as those of the original mutants (icmB and icmB/pMW100 in Fig. 4). These results may support the notion that the icm-dot apparatus is not directly involved in the transfer of chromosomal DNA.
In addition to the icm-dot system, L. pneumophila has another type IV secretion system designated the lvh (Legionella vir homolog) system (26). The lvh system is dispensable for intracellular growth in human macrophages and A. castellanii but is able to transfer a mobilizable IncQ plasmid (26). To examine whether the lvh system is involved in the conjugal transfer of chromosomal DNA, conjugation experiments were performed with lvh deletion strains as donors, that is, LELA2883-28 (dotB lvh) and LELA4432-28 (icmE lvh) (Table 1). As shown in Fig. 4, both strains transferred their chromosomes as well as the original mutant strains did. The frequencies of spontaneous mutation of LELA2883-28 and LELA4432-28 to Rifr were (1.0 ± 1.2) × 10−7 and (4.9 ± 2.9) × 10−7, respectively. This finding shows that the lvh system is dispensable for the transfer of chromosomal DNA. Therefore, there must be additional conjugal transfer systems in L. pneumophila. Recently, we found the possibility of a third conjugation system separate from icm-dot and from lvh on the L. pneumophila chromosome (the right end of contig 619 [CTG. WG. 013. 49. WK1. 091001]). The cluster consists of hits to TraD, -G, -H, -F, -N, -U, -W, -C, and -B, which are involved in sex pilus assembly (http://genome3.cpmc.columbia.edu/∼legion/index.html). Further studies are required to reveal mechanisms or apparatuses of chromosomal transfer.
In the present study, we showed that L. pneumophila virulence conversion occurs by the conjugal transfer of chromosomal DNA. The chromosome conjugation system is not specific for the serogroups or the strains used in this study because some transconjugants were obtained with L. pneumophila strains Bloomington-2 (serogroup 3, ATCC 33155) and AM240 (serogroup 1) (11; unpublished observation). As far as we know, this is the first report on virulence conversion by conjugal transfer of chromosomal DNA. The results of this study indicate that an avirulent strain could change to a virulent one through conjugal transfer from another virulent strain coinhabiting the environment. It is known that L. pneumophila is recovered from biofilms on the surfaces of water systems (3, 20, 29). The relative spatial stability of bacteria in biofilms may be a good niche for conjugation. It was reported that a high frequency of plasmid conjugation from Escherichia coli to Alcaligenes eutrophus occurs in biofilms (7). We are currently investigating whether L. pneumophila chromosomal conjugation occurs in biofilms. That study may provide new insights into the ecology of L. pneumophila and reveal the consequences of chromosomal conjugation in L. pneumophila.
Acknowledgments
This work was supported in part by National Institutes of Health grant AI-23549 to H.A.S.
We thank Laura Hales, Gil Segal, John Chen, and Yasuo Mizuguchi for helpful discussions and technical advice. We also thank Carmen Rodriguez and Kazuki Yamauchi for laboratory maintenance.
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