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Molecular Microbiology (1999) 34(4), 799±809 Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila Gil Segal,1² James J. Russo2 and Howard A. Shuman1* 1 Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA. 2 Columbia Genome Center, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, New York, NY 10032, USA. Summary We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another. Introduction Legionella pneumophila, the causative agent of Legionnaire's disease, is a facultative intracellular pathogen. Received 18 June, 1999; revised 20 August, 1999; accepted 25 August, 1999. ²Present address: Department of Molecular Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel. *For correspondence. E-mail [email protected]. columbia.edu; Tel. (1) 212 305 6913; Fax (1) 212 305 7323. Q 1999 Blackwell Science Ltd L. pneumophila is able to infect, multiply within and kill human macrophages, as well as free-living amoebae (Horwitz and Silverstein, 1980; Rowbotham, 1980). The bacteria are taken up by regular phagocytosis or by a special mechanism termed `coiling' phagocytosis (Horwitz, 1984; Bozue and Johnson, 1996). The bacteria are then found within a specialized phagosome that does not fuse with lysosomes or acidify (Horwitz, 1983a; Horwitz and Max®eld, 1984; Bozue and Johnson, 1996). The specialized phagosome undergoes several recruitment events that include association with smooth vesicles, mitochondria and rough endoplasmic reticulum (Horwitz, 1983b; Swanson and Isberg, 1995; Abu Kwaik, 1996). The bacteria multiply within the specialized phagosome until the cell eventually lyses, releasing bacteria that can start new rounds of infection (Horwitz and Silverstein, 1980; Rowbotham, 1980). Two regions of genes required for human macrophage killing and intracellular multiplication have been discovered in L. pneumophila (reviewed in Segal and Shuman, 1998a; Vogel and Isberg, 1999). Region I contains seven genes (icmV, -W and -X, and dotA, -B, -C and -D ) (Marra et al., 1992; Berger et al., 1994; Brand et al., 1994; Vogel et al., 1998), and region II contains 17 genes (icmT, -S, -R, -Q, -P, -O, -N, -M, -L, -K, -E, -G, -C, -D, -J, -B and -F ) (Segal and Shuman, 1997; Andrews et al., 1998; Purcell and Shuman, 1998; Segal et al., 1998; Vogel et al., 1998). Most of these genes were also shown to be required for intracellular growth in the protozoan host Acanthamoeba castellanii (Segal and Shuman, 1999a). Fourteen of these Icm/Dot proteins (IcmT, -P, -O, -L, -K, -G, -C, -D, -J, -B and DotA, -B, -C and -D) were found to contain signi®cant sequence similarity to Tra/Trb proteins from the IncI plasmid colIb-P9 (Segal and Shuman, 1999b), and IcmE was found to contain signi®cant sequence similarity to the TrbI protein from the IncP plasmid RK2 (Segal et al., 1998). Two of the Icm/Dot proteins (IcmE and DotB) were found to contain weak sequence similarity to two proteins that belong to the Agrobacterium tumefaciens vir type IV secretion system (VirB10 and VirB11 respectively; Vogel et al., 1998) involved in T-DNA transfer into plant cells. In addition to the sequence homologies, it was shown that L. pneumophila can conjugate RSF1010-related plasmids in an icm/dot-dependent manner (Segal and Shuman, 1997; Segal et al., 1998; Vogel et al., 1998), and an active 800 G. Segal, J. R. Russo and H. A. Shuman RSF1010 conjugation system was shown to inhibit intracellular growth and human macrophage killing (Segal and Shuman, 1998b). The A. tumefaciens vir type IV secretion system was also shown to conjugate RSF1010-related plasmids, and RSF1010 conjugation was shown to inhibit T-DNA transfer into plant cells, and result in the attenuation of virulence (Beijersbergen et al., 1992; Binns et al., 1995). At the present time, several type IV secretion systems have been identi®ed in bacteria and plasmids, among which the best studied is the vir system of A. tumefaciens (reviewed in Christie, 1997; Zupan et al., 1998; Burns, 1999). In addition to transfer of T-DNA into plant cells, the A. tumefaciens vir system was shown to transfer other substrates, such as the VirE2 protein, the VirF protein and RSF1010-related plasmids (Beijersbergen et al., 1992; Regensburg-Tuink and Hooykaas, 1993; Binns et al., 1995). Other bacteria and plasmids that contain a type IV secretion system include the pertussis toxin ptl exporter of Bordetella pertussis and the tra conjugative system of the IncN plasmid, pKM101, and the IncW plasmid, R388 (Weiss et al., 1993; Pohlman et al., 1994; Rivas et al., 1997). More recently, proteins homologous to type IV secretion systems have also been found in the Helicobacter pylori cag pathogenicity island and in Rickettsia prowazekii. The substrates secreted by these two systems are not known, but they are believed to be involved in the pathogenesis of these bacteria (Covacci et al., 1997; Andersson et al., 1998; Covacci et al., 1999). The aim of the study presented here is to characterize a newly discovered L. pneumophila type IV secretion system (lvh ) that is distinct from the icm/dot system which is required for intracellular growth in human macrophages and amoebae. One hypothesis is that the lvh system is required for intracellular growth in human macrophages and/or amoebae. A second hypothesis is that it is not required for intracellular growth. The results presented here clearly show that the lvh system is dispensable for intracellular growth in both these hosts. However, the lvh system was found to be partially required for RSF1010 conjugation, and it can substitute for some components of the icm/dot system in plasmid conjugation. macrophages and amoebae) and the A. tumefaciens vir system (required for T-DNA transfer to plant cells) share functional similarities and sequence homologies. First, both systems were shown to conjugate RSF1010-related plasmids between bacteria, using the icm/dot or vir gene products respectively. Second, the virulence of both systems was shown to be inhibited by an active RSF1010 conjugation system. Third, both systems contain proteins homologous to plasmid-encoded proteins involved in conjugation. Last, the two systems share weak sequence similarity between each other for two proteins (the C-terminal region of IcmE is homologous to VirB10, and DotB is homologous to VirB11; summarized in Segal and Shuman, 1998a; Vogel and Isberg, 1999). Due to these functional similarities and sequence homologies, together with the known involvement of type IV secretion systems in bacterial virulence, we decided to characterize the L. pneumophila VirB4 and VirD4 homologues. Cloning of the DNA region containing the L. pneumophila vir homologues DNA fragments containing the L. pneumophila virB4 and virD4 homologues were ampli®ed by polymerase chain reaction (PCR) and cloned. The cloned DNA fragments were used to screen an L. pneumophila cosmid library and several positive cosmids were identi®ed. Restriction digests of these cosmids revealed that the two vir homologues are located about 7 kb from one another on the same 23 kb EcoRI fragment. The nucleotide sequence was determined for the region between the virB4 and virD4 homologues and, after potential open reading frames (ORFs) were identi®ed, additional sequence information was obtained to cover all the vir homologues and several ORFs downstream and upstream from the vir homologous region. The ORFs found in this region that are homologous to the A. tumefaciens Vir proteins were named lvh for Legionella vir homologues (the last letter of the name and the number are identical to the A. tumefaciens vir genes). Eleven ORFs homologous to A. tumefaciens Vir proteins were found, with the order lvhB2-B3-B4-B5-B7-B6-B8-B9B10-B11-D4 (Fig. 1). Results In the preliminary phase of a joint project of the Columbia Genome Center and our laboratory aimed at sequencing the entire L. pneumophila genome, several hundred random sequences of about 500 bp were generated. When a BLAST search was performed on these DNA sequences and the protein sequences they were predicted to encode, two of the protein sequences were found to be homologous to the A. tumefaciens VirB4 and VirD4 virulence proteins. Recently, it has been shown that the L. pneumophila icm/ dot system (required for intracellular growth in human The L. pneumophila vir homologues are located on a DNA island DNA analysis of the region containing the lvh genes revealed a higher GC content (44%) in comparison with the known GC content of the L. pneumophila chromosome (39%) (Brenner et al., 1978; Segal et al., 1998). The borders of the high GC region were found to be located between ORFs. In the upstream region, the border was located between two ORFs pointing in the opposite orientation, and in the downstream region between two ORFs pointing Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 Legionella type IV secretion system 801 Fig. 1. Schematic representation of genes belonging to type IV secretion systems. Genes are represented as boxes with arrowheads indicating their orientation and are identi®ed by the fourth letter (and the number if used) of their standard designation (the ®rst three or two letters are indicated to the left). The homologous genes are shaded in the same pattern in all the systems. Genes not homologous to type IVrelated genes are not shaded. Only the part of the protein homologous to proteins of type IV secretion systems is shaded. The type IV-related genes of R. prowazekii are found on two separated regions on the chromosome. The type IV-related genes of H. pylori are found on three separated regions on the chromosome, one of them in the cag pathogenicity island. in the same direction. In addition to the 11 vir homologues found in the high GC region, ®ve other ORFs, not homologous to the A. tumefaciens Vir proteins, were found in it and named lvr for Legionella vir region (lvrA to lvrE ). The lvrC gene, located upstream of lvhB2, was found to be homologous to the Escherichia coli carbon storage regulator gene, csrA (38.8% identity, 53.7% similarity, over the whole length of the protein). It is interesting to note that, in the A. tumefaciens vir region, the virA gene is located upstream of the virB operon. The virA and virG genes (virG is located downstream of the virB operon) encode a two-component regulatory system that controls vir gene expression. It is possible that, in a similar manner to virA, the csrA homologue controls the expression of the lvh genes located downstream of it. The other Lvr proteins (LvrA, B, D and E) contain no sequence similarity to any known sequences found in GenBank. Only one of the lvr genes (lvrD ) was found to be located in the middle of the lvh genes, between lvhB7 and lvhB6 (Fig. 1). The order of the vir homologues near lvrD is different from the one found in other type IV secretion systems, as lvhB7 is located upstream of lvhB6 (Fig. 1). A region containing lower GC content (37%) then the L. pneumophila Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 chromosome (39%) was found upstream of the high GC region. The ORF located in this region was found to be homologous to the lambda repressor cII (24.8% identity, 35.2% similarity, over the whole length of the protein). The ORF located downstream of the high GC region shares no sequence homology with any genes or proteins found in GenBank. Sequence comparison with other type IV secretion systems Several bacteria and plasmids have been shown to contain type IV secretion systems, among which the A. tumefaciens vir system is the best studied (for review see Christie, 1997; Zupan et al., 1998). All of these systems contain homologues to several of the A. tumefaciens vir genes, and the gene arrangement is similar to the one found in A. tumefaciens (Fig. 1). Only six Vir-related proteins (VirB4, -B7, -B8, -B9, -B10 and -B11) were found to be present in all the bacteria and plasmids that were shown to contain a type IV secretion system. The VirD4 protein has also been found in all the systems except the B. pertussis ptl system. It may be present in this system as well, but not 802 G. Segal, J. R. Russo and H. A. Shuman yet identi®ed due to a lack of suf®cient sequence information. When a sequence comparison of the L. pneumophila Lvh proteins with all the other known proteins of type IV secretion systems was performed, we found that from the seven conserved proteins (VirB4, -B7, -B8, -B9, -B10, -B11 and -D4) of type IV secretion systems, four of the L. pneumophila Lvh proteins (LvhB4, -B9, -B11 and -D4) contain the highest degree of homology to their homologues from the type IV secretion system of the human obligate intracellular pathogen R. prowazekii. The fact that these two bacteria are human intracellular pathogens might indicate a special adaptation of the system to the intracellular environment. The lvh genes are dispensable for intracellular growth in HL-60-derived human macrophages and A. castellanii To test if the lvh genes are involved in the intracellular life style of L. pneumophila, two strains containing a complete deletion of the lvh region were constructed. Both strains (GS-28G and GS-28K) were tested for their ability to grow inside HL-60-derived human macrophages and the protozoan host A. castellanii. The results of an intracellular growth experiment are shown in Fig. 2. No defect was observed in the ability of these mutants to grow intracellularly in either host. We conclude that in an otherwise wildtype background the lvh gene products are not required for intracellular multiplication in either host. We also tested the same strains for their abilities to kill HL-60-derived macrophages. Again, we observed no differences between the strains containing the complete lvh deletion and the wildtype control strain with respect to the ability to kill HL-60derived macrophages (data not shown). We also asked if the intracellular multiplication or macrophage killing defects of icmE and dotB mutants could be restored by a plasmid carrying the lvh genes, but found no effect of the plasmid on these strains (data not shown). We conclude that the lvh gene products do not play an essential role in the ability of L. pneumophila to grow within or kill human macrophages or A. castellanii. The lvh genes are partially required for RSF1010 conjugation In previous reports, we showed that RSF1010-related plasmids can conjugate between two L. pneumophila strains, and conjugation was shown to be dependent on several icm/dot genes (Segal and Shuman, 1998b; Segal et al., 1998). Compared with wild-type bacteria, strains with mutations in several icm/dot genes exhibited signi®cant defects in the ability to transfer the IncQ plasmid, pMMB207, to L. pneumophila recipient cells. In some cases the defect was severe, whereas in others moderate transfer frequencies were retained. We suggested that these results could be explained either by a partial requirement for some dot/icm components, or alternatively by the presence of another conjugal system whose components might substitute for the missing dot/icm gene product (Segal and Shuman, 1998b; Segal et al., 1998). It is known that plasmids, such as R388 and pKM101, that contain a type IV secretion system use this system for their conjugation (Winans and Walker, 1985; Rivas et al., 1997). This information led us to test whether the L. pneumophila lvh type IV secretion system is involved in conjugation of RSF1010-related plasmids. The strain containing a complete deletion of the lvh region (GS-28K) was found to have a 10-fold reduction in conjugation frequency in comparison with the wild-type strain when tested as donor (Fig. 3A). This reduction in conjugation frequency was complemented to the wild-type level when a plasmid containing the lvh region was introduced into this strain (Fig. 3A). It is probable that the relatively high conjugation frequency found in the lvh mutant is a result of conjugation activity of the icm/dot system. Fig. 2. Intracellular growth of lvh deletion mutants in A. castellanii and HL-60-derived macrophages. Intracellular growth experiments in A. castellanii (A) and HL-60derived macrophages (B), were carried as described in the Experimental procedures section. The experiments were done at least three times and similar results to those shown were obtained. Strains tested (see Table 1): JR32, B; 25D, l; GS-28G, K; GS-28K, W. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 Legionella type IV secretion system Fig. 3. Conjugation properties of icm/dot and lvh insertion mutants. Different icm/dot and lvh insertion mutants, containing pMMB207ab or complementing plasmids, were used as donors. A. Conjugation analysis of the lvh mutant. B. Conjugation analysis of the dotB±lvh double mutant. C. Conjugation analysis of the icmE±lvh double mutant. The scale in part A is different from the scale in parts B and C. The icmT insertion mutant (LELA4086R) was used as a recipient in all the matings. The donor strains tested were wild-type JR32, LELA4432 for icmE, LELA2883 for dotB, GS-28K for lvh, LELA4432-28 for icmE±lvh and LELA2883-28 for dotB±lvh. The complementing plasmids used were pGS-Lc-47 for icmE, pGS-VBD-32 for lvh and pGS-BCD-04 for dotB. Characterization of double mutants in icm/dot and lvh genes for RSF1010 conjugation The icm/dot system contains two proteins homologous to proteins of type IV secretion systems (IcmE and DotB are homologous to VirB10 and VirB11 respectively). Strains containing insertion mutations in these two genes Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 803 (LELA4432 for icmE and LELA2883 for dotB ) were found to be moderately attenuated for RSF1010 conjugation (Fig. 3B and C). We were interested to know whether the residual conjugation activity is a result of the presence of the lvh type IV secretion system compensating for the missing icm/dot gene products. To test this hypothesis, we introduced a complete deletion of the lvh region to strains containing an insertion in icmE (LELA4432) and dotB (LELA2883). The conjugation frequencies of the two double mutants (LELA4432-28 for icmE ±lvh and LELA2883-28 for dotB ±lvh ) were dramatically reduced in comparison with the single insertion in each of these genes (Fig. 3B and C). These results indicate that the lvh genes are responsible for the residual conjugation activity observed with the original icmE and dotB insertion mutants. Plasmids containing either the dotBCD genes (pGSBCD-04) or the entire lvh region (pGS-VBD-32) (Fig. 3B) were able to complement the conjugation phenotype of the dotB±lvh double mutant. The conjugation frequencies of the complemented mutants were close to those of the dotB or lvh mutants (LELA2883 and GS-28K respectively). Both these plasmids (pGS-VBD-32 and pGS-BCD-04) did not, however, complement the weak reduction in conjugation frequency observed with the original dotB insertion mutant (Fig. 3B). The dotB±lvh double mutant exhibited a very low level of conjugation (Fig. 3B and C, compare the dotB±lvh and the icmE±lvh double mutants). This activity might be due to yet other virB11-related gene products. It is known that L. pneumophila contains at least three members of the VirB11 protein family: DotB in the icm/ dot system; LvhB11 in the newly described type IV secretion system; and PilB in the pilin biosynthesis system (Liles et al., 1998). A fourth member is expected to be found as part of a type II secretion system that was found in L. pneumophila (Hales and Shuman, 1999). One or both of the other two members of this protein family might be responsible for the very low level of RSF1010 conjugation observed in the absence of the dotB and lvhB11 gene products. The icmE±lvh double mutant (LELA4432-28) was found to be completely defective for conjugation. This mutant was complemented close to the conjugation levels of the icmE or lvh mutants (LELA4432 and GS-28K respectively), with a plasmid containing the whole lvh region (pGS-VBD-32) (Fig. 3C). A very low level of complementation was observed when a plasmid containing the icmE gene (pGS-Lc-47) was introduced into this strain (Fig. 3C). The reason for the weak complementation is not known, but this plasmid complemented the original icmE insertion mutant (LELA4432) for conjugation (Fig. 3C), and partially complemented it for intracellular growth (Segal et al., 1998), indicating that the icmE gene product is expressed from this plasmid. A interesting result was obtained when we tested the 804 G. Segal, J. R. Russo and H. A. Shuman conjugation frequency of the icmE mutant (LELA4432) with a plasmid containing the lvh genes (pGS-VBD-32). A reduction of about 500-fold in conjugation frequency was observed with this plasmid in comparison with the vector (Fig. 3C). This is not due to the size difference between the two plasmids (about 30 kb in comparison with 9 kb), as these two plasmids had a similar conjugation frequency when tested in the wild-type strain (Fig. 3A). The reason for the reduction in conjugation frequency is not known, but might result from complex relations between the icm/ dot and lvh systems that are sensitive to changes in the stoichiometries of the components. We did not perform a similar analysis with the icmE and dotB insertion mutants and the icmE±lvh and dotB±lvh double mutants for intracellular growth, because the original icmE and dotB mutants by themselves are completely unable to grow inside HL-60-derived human macrophages and A. castellanii (Segal and Shuman, 1999a; data not shown). Discussion L. pneumophila is a broad host range facultative intracellular pathogen that overcomes many natural host defence mechanisms, enabling it to cause disease in man. Like Mycobacterium tuberculosis (Armstrong and D'Arcy Hart, 1971), Chlamydia psittaci (Friis, 1972) and Toxoplasma gondii (Jones and Hirsch, 1972), L. pneumophila multiplies within human cells inside a specialized vacuole that does not fuse with secondary lysosomes (Horwitz, 1983a). In nature, L. pneumophila uses a similar mechanism to infect and multiply within free-living amoebae (Abu Kwaik, 1996; Bozue and Johnson, 1996; Fields, 1996). During the past few years, 24 icm/dot genes have been described in L. pneumophila. These genes have been shown to be speci®cally required for intracellular growth in human macrophages and amoebae (Marra et al., 1992; Berger et al., 1994; Brand et al., 1994; Segal and Shuman, 1997; Andrews et al., 1998; Purcell and Shuman, 1998; Segal et al., 1998; Vogel et al., 1998; Segal and Shuman, 1999a). Fourteen proteins encoded by these genes were shown to display homology with proteins involved in conjugation from the IncI plasmid colIb-P9 (Segal and Shuman, 1999b). In addition, two of these proteins (IcmE and DotB) were shown to contain weak homology to the A. tumefaciens Vir proteins (VirB10 and VirB11) that are part of a type IV secretion system. Interestingly, both the A. tumefaciens vir type IV secretion system and the L. pneumophila icm/dot virulence system were shown to mediate conjugation of RSF1010-related plasmids between bacteria (Beijersbergen et al., 1992; Segal et al., 1998; Vogel et al., 1998), and both systems were shown to be inhibited by an active RSF1010 conjugation system (Binns et al., 1995; Segal and Shuman, 1998b). These ®ndings suggest that the icm/dot system might be involved in transferring an effector molecule to the host cell during infection, in a similar manner to the transfer of T-DNA into plant cells during A. tumefaciens infection. Here, we describe a new L. pneumophila type IV secretion system (lvh ), which is distinct from the icm/dot system. This system contains 11 of the 12 genes (missing VirB1) that were shown to be required for T-DNA transfer and RSF1010 conjugation in A. tumefaciens (VirB1 was shown to be only partially required for T-DNA and RSF1010 transfer; Fullner, 1998). We found that the lvh system is dispensable for intracellular growth of L. pneumophila in both HL-60-derived human macrophages and A. castellanii. Currently, there is no information regarding the possibility that this system is required for intracellular growth of L. pneumophila in other hosts, such as Naegleria or Tetrahymena (Fields et al., 1984; Newsome et al., 1985). In addition to the well-characterized A. tumefaciens vir type IV secretion system, several other type IV secretion systems have been described in bacteria and plasmids, and they are summarized in Fig. 1. Type IV secretion systems share many homologous proteins, and other multicomponent systems have also been shown to contain homologues to proteins of type IV secretion systems (Whitchurch et al., 1991; Lessl et al., 1992; Hobbs and Mattick, 1993). The components of all known type IV secretion systems, together with representatives from other systems in which homologues to proteins of type IV secretion systems have been found, are summarized in Fig. 4. The trb system, involved in mating pair formation of the plasmid RK2 and the A. tumefaciens Ti plasmid (the same plasmid on which the vir type IV secretion system is located) contains several homologues to proteins of type IV secretion systems (Li et al., 1998). In addition, type II secretion systems and pilin biosynthesis systems contain one homologue to a protein from type IV secretion systems (VirB11). A homologue to this protein was also found in the icm/dot system (dotB ), together with a VirB10 homologue (IcmE). The homology of the DotB and IcmE proteins to proteins of type IV secretion systems suggested the idea that the icm/dot system might resemble a type IV secretion system (Vogel et al., 1998). However, the recent ®nding that 14 Icm/Dot proteins (including DotB) share signi®cant sequence similarity with 14 conjugation-related proteins from the IncI plasmid colIb-P9 strongly suggests that the origin of the icm/dot system is from the transfer region of an IncI plasmid and not from a type IV secretion system (Segal and Shuman, 1999b). When we compared the homology of DotB and LvhB11 with other members of their protein family, it became clear that DotB is more closely related to proteins that are part of the colIb-P9 tra/trb system and pilin biosynthesis systems, and that LvhB11 is more homologous to proteins that belong to type IV secretion systems. A similar result was obtained when Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 Legionella type IV secretion system 805 Fig. 4. Homologues of proteins from type IV secretion systems present in other bacterial multicomponent systems. The upper box indicates the secretion system in which homologues were found. Type IV, type IV secretion system; Mpf, mating pair formation; Type II, type II secretion system; Pili, pilin biosynthesis. The second box from the top shows the bacteria or plasmid system presented (all the known type IV secretion systems are shown, together with two representatives from each of the other systems). Agr, Agrobacterium tumefaciens ; Leg, Legionella pneumophila ; Bor, Bordetella pertussis ; R388, the IncW plasmid R388; 101, the IncN plasmid pKM101; Ric, Rickettsia prowazekii ; Hel, Helicobacter pylori ; RK2, the IncP plasmid RK2 (RP4); Kle, Klebsiella oxytoca ; Erw, Erwinia chrysanthemi ; Pse, Pseudomonas aeruginosa ; Nei, Neisseria gonorrhoeae ; colIb, the IncI plasmid colIb-P9. The gene name and the number of components that are known to be part of each system are written under the bacterial or plasmid name. In the left column, the last letter and number of the A. tumefaciens vir gene nomenclature is given. *, located in another region of the genome; ^, an estimate from the number of vir homologues found; #, some of the genes are located outside of the cag pathogenicity island. we compared the homology of IcmE and LvhB10 with other members of their protein family. IcmE was found to be more closely related to a protein that is part of the RK2 plasmid mating pair formation system and less related to proteins from type IV secretion systems. As can be seen in Fig. 4, VirB11 homologues are found in many systems involved in secretion. With the exception of IcmE, VirB10 homologues were found only in type IV secretion systems and in systems involved in mating pair formation. IcmE is the only protein in the Icm/Dot system that has a homologue in the IncP plasmid RK2 (TrbI), but does not have a homologue in the colIb-P9 plasmid. In addition, IcmE has been shown to contain a unique sequence feature, a multiple repeat of 10 amino acids in the middle of the protein between a predicted transmembrane domain and the VirB10 homologous region (Fig. 5). We were delighted to ®nd a very similar sequence feature in the H. pylori VirB10 homologue, Cag7 (Fig. 5), located in the type IV secretion system of the cag pathogenicity island (Fig. 1). Other VirB10 homologues, including the Fig. 5. Dot plot analysis of the L. pneumophila IcmE protein and the H. pylori Cag7 protein. The analysis shows the multiple repeats present in the middle sections of these two proteins. The regions predicted to form a transmembrane domain and the region homologous to VirB10 proteins are marked. The numbers on both axes indicate amino acid positions. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 806 G. Segal, J. R. Russo and H. A. Shuman L. pneumophila LvhB10 protein, do not contain this unique sequence feature. This ®nding might indicate a mechanistic similarity between the cag pathogenicity island type IV secretion system and the icm/dot system. Taking all this information together, we believe that the icm/dot system is not a type IV secretion system. The icm/dot system is most closely related to the conjugation system of IncI plasmids, and may represent a new type of secretion system that contains homologous proteins to other secretion systems. Even though the icm/dot system is not closely related to type IV secretion systems, the analysis of double mutants in the lvh system and the icm/dot system has revealed interesting relationships. The original strains, containing an insertion in the icmE and dotB genes (these genes encode for the only two proteins in the icm/dot system that contain homology to proteins from type IV secretion systems), have a small reduction in conjugation frequency, but they are completely defective for intracellular growth. When the lvh region was deleted in the background of these insertions, conjugation was almost abolished. These results indicate that in the dotB and icmE insertion mutants components of the lvh type IV secretion system can substitute for their function during conjugation, but not for intracellular growth. Taking together three lines of evidence, showing that (i) the lvh system can replace some of the icm/dot components for conjugation, but not for intracellular growth, (ii) the type IV lvh system by itself cannot carry out conjugation (as indicated by the fact that insertions in several icm/dot genes completely abolished conjugation; Segal et al., 1998; Vogel et al., 1998), and (iii) an active RSF1010 conjugation system can inhibit intracellular growth by L. pneumophila (Segal and Shuman, 1998b), leads to at least two models that can explain the inhibition of intracellular growth by an active RSF1010 conjugation system. The ®rst model (Segal and Shuman, 1998b) suggests that the MobA protein competes with as yet unknown effector proteins for the Icm/Dot transfer apparatus. A second model is suggested by the new results described here, indicating that icm/dot components can participate in the formation of more then one kind of complex. In this model, the presence of the MobA protein results in the formation of an abnormal Icm/Dot transfer complex that can conjugate, but can not participate in intracellular growth. These conjugation complexes might resemble those formed when Icm/Dot components interact with Lvh components to form a complex that can participate in conjugation but can not promote intracellular growth. L. pneumophila probably contains at least four homologues of the VirB11 protein (DotB in the icm/dot system, PilB in the pilin biosynthesis system, LvhB11 in the newly described type IV secretion system and a fourth homologue in the type II secretion system). All these systems are known to be multicomponent systems that involve complex protein±protein interactions. As we have shown here, interactions can occur not only between proteins that belong to the same system, but also between proteins that belong to different systems. Appropriate interactions within and among these systems are likely to be important for the proper function of these multicomponent systems. In addition, novel combinations of related components from different systems can generate new functions. Experimental procedures Bacterial strains, plasmids and media Bacterial strains and plasmids used in this work are described in Tables 1 and 2 respectively. Bacterial media, plates and antibiotic concentrations were used as has been described previously (Segal and Shuman, 1997). Gentamicin (Gm) was used at a concentration of 10 mg ml 1 for L. pneumophila, and 25 mg ml 1 for E. coli. Plasmid construction for complementation The DNA sequences generated by the Columbia Genome Center that contain part of the L. pneumophila virB4 and virD4 homologues, were used to design primers for PCR. The primers 58-GCTTTTATTTCGGGGAGTGC-38 and 58GTTTCCTCCGAAAATGGCGG-38 were used to amplify a 677 bp fragment of the virB4 homologue (3771±4448 in the sequence), and the primers 58-CATAGGCTTTACCGATGATG-38 and 58-AGACTCGGAATAACCTTGGG-38 were used to amplify a 414 bp fragment of the virD4 homologue Table 1. L. pneumophila strains. Strains Genotype and features Reference or source 25D GS-28K GS-28G JR32 LELA2883 LELA2883-28 LELA4432 LELA4432-28 LELA4086R Icm avirulent mutant JR32 with Km insertion instead of the lvh region JR32 with Gm insertion instead of the lvh region Salt-sensitive isolate of AM511 JR32 dotB2883 ::Tn903 dIIlacZ LELA2883 with Gm insertion instead of the lvh region JR32 icmE4432 ::Tn903 dIIlacZ LELA4432 with Gm insertion instead of the lvh region Rifampicin derivative of LELA4086 Horwitz (1987) This study This study Sadosky et al. (1993) Sadosky et al. (1993) This study Sadosky et al. (1993) This study Segal and Shuman (1998b) Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 Legionella type IV secretion system 807 Table 2. Plasmids used in this study. Plasmid Features Reference or source pGS-BCD-04 pGS-cos-04 pGS-VBD-c1 pGS-VBD-2 pGS-VBD-10 pGS-VBD-11 pGS-VBD-28 pGS-VBD-28-Gm pGS-VBD-28-Gm-GR pGS-VBD-28-Km pGS-VBD-28-Km-GR pGS-VBD-32 pGS-VirB4 pGS-VirD4 pLAW344 pMMB207ab pUC-18 dotBCD in pMMB207ab dotABCD, icmVWX in pLAFR1 The entire lvh region in pLAFR1 lvhB4,B5,B7,B6,B8,B9,B10 in pUC-18 lvrAB and 4 kb upstream region in pUC-18 Part of lvhB4 in pUC-18 Part of lvhD4 in pUC-18 pGS-VBD-28 with Gm insertion instead of the lvh genes The insert of pGS-VBD-28-Gm in pLAW344 pGS-VBD-28 with Kminsertion instead of the lvh genes The insert of pGS-VBD-28-Km in pLAW344 The entire lvh region in pMMB207ab Part of lvhB4 in pUC-18 Part of lvhD4 in pUC-18 sacB MCS oriT(RK2) Cm r loxP oriR(colEI) Apr loxP pMMB207 containing MCS (a-complementation) oriR(colEI) MCS Apr This study G. Segal and H. A. Shuman (unpublished) This study This study This study This study This study This study This study This study This study This study This study This study Wiater et al. (1994) Segal and Shuman (1997) Yanish-Perron et al. (1985) (12 597±13 011 in the sequence). Both fragments were cloned into the HincII site of pUC-18 to generate pGS-VirB4 and pGS-VirD4. The inserts of these plasmids were used as probes for colony hybridization (Segal and Ron, 1993), with the L. pneumophila pLAFR1 cosmid library (Szeto and Shuman, 1990). Several positive cosmids were identi®ed and the same 23 kb EcoRI fragment was present in all of them (as well as some other EcoRI fragments). All the cosmids hybridized with both probes. One of these cosmids (pGSVBD-c1) was used for all the subclones described below. To construct complementing plasmids for the genes found in the lvh region, the 23 kb EcoRI fragment was subcloned from pGS-VBD-c1 into pMMB207ab to generate pGS-VBD-32. The plasmid pGS-BCD-04 was constructed by the subcloning of a 10 kb BamHI fragment from the cosmid pGS-cos04, containing icm/dot region I (G. Segal and H. A. Shuman, unpublished), into pMMB207ab. This plasmid contained the dotBCD genes, and it was used for complementation of the dotB insertion mutant LELA2883 and the dotB±lvh double mutant LELA2883-28. Plasmid construction for allelic exchange Two plasmids, containing drug resistance elements in place of the lvh genes, were constructed for allelic exchange. To generate a complete deletion of the lvh region, an upstream 3.5 kb HindIII±Pst I fragment (an upstream HindIII site and the Pst I site at 2131 in the sequence) was cloned into pUC-18 to generate pGS-VBD-10. A second, downstream 3.5 kb XbaI±SacI fragment (the XbaI site at 14 370 in the sequence and a downstream SacI site) was cloned into this plasmid to generate pGSVBD-28. This plasmid contains a unique Sal I site between the two inserts (the origin of this Sal I site is from the pUC-18 polylinker). The Km resistance cassette was cloned into this Sal I site to generate pGS-VBD-28-Km. This plasmid, containing the Km resistance cassette instead of all the lvh genes, was digested with PvuII, and the insert was cloned into the EcoRV site of the allelic exchange vector pLAW344. The plasmids generated were used for allelic exchange, as has been described previously (Segal and Shuman, 1997). We were unable to use pGS-VBD-28-Km to generate double Q 1999 Blackwell Science Ltd, Molecular Microbiology, 34, 799±809 mutants of the lvh genes and the icm/dot genes because the icm/dot insertion mutants are already Km resistant. To overcome this problem, we cloned the Gm resistance cassette into the XbaI site (after ®ll-in) of pGS-VBD-28 to generate pGSVBD-28-Gm. This plasmid was digested with HindIII, ®lled in and cloned into the EcoRV site of the allelic exchange vector pLAW344. Allelic exchange was performed as has been previously described (Segal and Shuman, 1997), but Gm was used for selection instead of Km during the procedure. Bacteria and host assays The following assays were performed as has been previously described: (i) intracellular growth in HL-60-derived macrophages (Segal and Shuman, 1999a); (ii) intracellular growth in A. castellanii (Segal and Shuman, 1999a); and (iii) L. pneumophila mating (Segal and Shuman, 1998b). DNA sequencing and sequence analysis The sequence was generated by the Columbia Genome Center and the DNA Synthesis and Sequencing Facility of the Comprehensive Cancer Center, College of Physicians and Surgeons of Columbia University. Plasmid subclones of pGS-VBD-c1 were end sequenced using big dye terminators (Perkin-Elmer). For the whole genome shotgun, L. pneumophila DNA was prepared as has been described previously (Segal and Ron, 1993). Subsequently, 25 mg of the DNA was sonicated, shotgun cloned into M13mp18, and sequenced as has been described previously for large-insert clones (Russo et al., 1996). Some of the sequence was con®rmed by comparison with shotgun sequences, derived from BAC clones with insert DNA spanning the lvh region (S. Kalachikov, J. J. Russo, S. Fischer and E. Cayanis, data not shown), using the GENE HUNTING TOOL package, a suite of linked programs compiled by Dr Peisen Zhang in the Columbia Genome Center Informatics section. Primer walking was used to provide complete and high-accuracy sequence coverage of the locus. Sequence data for the complete lvh locus have been assigned EMBL data accession number Y19029. DNA sequences and amino acid sequences of potential open reading frames were 808 G. Segal, J. R. Russo and H. A. Shuman compared with GenBank, EMBL and SWISSPROT, using the BLAST version 2 program (Zheng et al., 1998). Sequence alignment was carried out using the ALIGN program (http:/ /vega. igh.cnrs.fr/bin/align-guess.cgi) and the PHRAP program (http:/ / www.mbt.washington.edu/phrap.docs/phrap.html). Acknowledgements This work was supported by a grant from the NIH (AI23549) and the Columbia Genome Center. G. Segal was supported by the Stephen A. Morse fellowship from Departments of Microbiology and Medicine of Columbia University. We thank Mr Minchen Chien and Ms Anthi Georghiou for generating the random nucleotide sequence. We are grateful to Ms Carmen Rodriguez for excellent technical assistance during this work. References Abu Kwaik, Y. (1996) The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62: 2022±2028. 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