Download Relationships between a new type IV secretion system and the icm

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
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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
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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.
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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
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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.
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