Comparative sequence analysis of the icm/dot

mosomal loci. We demonstrate that these genes are present in all the L. pneumophila strains examined herein, but

1. Introduction

Legionella pneumophila, the causative agent of

Legionnaires disease, an occasionally fatal pneu-

monia, as well as much more common mild u-

like lung infections, is found in fresh waterthroughout the world. The Philadelphia 1 isolate

of L. pneumophila, named after the site of the

originally described outbreak in 1976 (Fraser

et al., 1977), is a member of the most prevalent

serogroup 1 (Fields et al., 2002). Isolates associ-

4) 12* Corresponding author. Fax: +212-851-5215.display a wide range of sequence variation among the dierent strains, none of which are clearly associated with vir-

ulence potential. The strains fall within seven phylogenetic groups, but discrepancies among the gene trees indicate a

complicated evolutionary history for the icm/dot loci, with perhaps two independent gene acquisition events and

subsequent genomic rearrangements. Signicant ndings include a probable t-SNARE domain in IcmG that may in-

dicate a direct role for this putative inner membrane protein in altering the hosts membrane fusion machinery, apotential functional domain in the central hydrophobic portion of IcmK that may allow it to participate in forming the

pore of the secretion complex, and strict conservation of the amino acid physicochemical characteristics in the IcmP

region corresponding to the trbA domain that could play a role in molecular transfer.

2004 Elsevier Inc. All rights reserved.

Keywords: Legionella pneumophila; icm/dot genes; Evolution; Phylogenetic analysis; Virulencegenes in Legionella

Irina Morozova,a,1 Xiaoyan Qu,a,1 Shundi Shi,a,1 Gifty Asamani,a

Joseph E. Greenberg,a Howard A. Shuman,b and James J. Russoa,*

a Columbia Genome Center, Columbia University College of Physicians and Surgeons, 1150 St. Nicholas Avenue,

New York, NY 10032, USAb Department of Microbiology, Columbia University College of Physicians and Surgeons, 701 W. 168th Street,

New York, NY 10032, USA

Received 18 August 2003, revised 21 November 2003

Abstract

The icm/dot genes in Legionella pneumophila are essential for the ability of the bacteria to survive within macro-

phages in lung infections such as Legionnaires disease, or amoebae in nature. The 22 genes of the complex, thought toencode a transport apparatus for transfer of eector molecules into the host cell cytoplasm, are located in two chro-Plasmid 51 (200E-mail address: jjr4@columbia.edu (J.J. Russo).1 These three authors contributed equally to the work.

0147-619X/$ - see front matter 2004 Elsevier Inc. All rights reservdoi:10.1016/j.plasmid.2003.12.0047147

www.elsevier.com/locate/yplasated with at least 15 other serogroups have been

ed.

described in the ensuing years (Helbig et al., 2002;Yu et al., 2002). In addition, L. pneumophila is one

of about 42 known species within the genus Le-

gionella (Fields et al., 2002; Yu et al., 2002), many

of which can be associated with clinical symptoms.

128 I. Morozova et al. / PlasmiAs part of its life cycle, Legionella bacteria are

taken up and survive within phagocytic cells (e.g.,

amoebae in the environment, macrophages in the

human lung). The bacteria replicate within intra-cellular vacuoles, and eventually kill the original

host cell, whereupon they may infect nearby

phagocytes (Swanson and Hammer, 2000). Among

other virulence genes, two regions with some fea-

tures of pathogenicity islands, the so-called icm/dot

gene clusters, appear to be essential for their

ability to survive within and kill macrophages and

amoebae (Andrews et al., 1998; Berger and Isberg,1993; Sadosky et al., 1993; Segal and Shuman,

1997; Vogel et al., 1998).

The icm/dot2 cluster I includes seven genes

(dotAD, icmV,W,X) and the larger cluster II

contains the remaining 17 members (icmT,S,R,

Q,P,O,N,M,L,K,E,G,C,D,J,B,F). Their encoded

proteins are thought to translocate eector mole-

cules into the host cell that somehow prevent thelatter from killing the proliferating bacteria,

probably by preventing phagosomelysosome fu-

sion in macrophages (Christie, 2001; Nagai et al.,

2002). The icm/dot loci are highly similar to the

transfer region of plasmid R64 and other IncI1

plasmids, and it has previously been suggested that

icm/dot virulence genes share a common ancestor

with plasmid conjugation genes (Komano et al.,2000; Segal and Shuman, 1999; Segal et al., 1998;

Sexton and Vogel, 2002). It is unclear if the icm/dot

genes derive from a single plasmid after which they

separated into the two gene clusters, or there were

multiple gene transfer events. Of the more than

100 bacteria for which complete genome sequence

is available, only Coxiella burnettii has homologs

of the full icm/dot genes; in Coxiella, all the icm/dotgenes are contained in a single locus (Seshadri

2 Many of these genes were discovered at about the same

time in two laboratories and referred to as either icm (intra-

cellular multiplication) or dot (defective in organellar track-

ing). Where a particular gene has two names, we use the icmdesignation in this paper.et al., 2003). Besides the icm/dot genes, Legionella,like many other bacteria, contain most members of

a Type IV secretion system, the lvh/lvr genes that

have virulence properties in some organisms,

though apparently not in L. pneumophila (Segal

et al., 1999).

Since most of the icm/dot proteins are clearly

implicated in the ability of Legionella to grow

and survive within macrophages, it would be ofinterest to know if any of them are missing or

considerably dierent in strains of lower patho-

genicity. In the present work, we demonstrated

the presence of several of the icm/dot genes in at

least seven L. pneumophila species and one or

more lvh/lvr genes in most of the Legionella species

tested.

The evolution of the icm/dot genes may be dis-tinct from the majority of the other Legionella

genes, particularly the housekeeping genes, either

because they are part of the bacterias virulencegene set, or because of their presumed plasmid

origin. Virulence genes are often subject to diver-

sifying selection and evolve faster than the rest of

the genome to avoid the hosts response to theinfection. But Legionella, with their largely intra-cellular lifestyle, are only briey exposed to the

mammalian immune system, and are not known to

establish infections in serial hosts; therefore they

are unlikely to undergo adaptive evolution. In-

deed, the mip gene, which encodes a possible vir-

ulence factor, was found to have relatively few

polymorphisms in L. pneumophila strains even

though it encodes an outer membrane protein(Bumbaugh et al., 2002). In addition, although the

quite variable dotA gene product was found to be

less conservative in its outer domains, the ratio of

synonymous and nonsynonymous nucleotide sub-

stitutions in this region did not indicate adaptive

evolution according to these same investigators

(Bumbaugh et al., 2002). In this study we ad-

dressed the question of whether the remaininggenes of the icm/dot loci show the same elevated

level of variability as dotA relative to other por-

tions of the genome, particularly housekeeping

genes.

A possible plasmid origin of the icm/dot genes

would account for dierences in evolutionary his-

d 51 (2004) 127147tory between these genes as a group and the rest of

Legionellas chromosomal genes. According to thishypothesis, the icm/dot loci would constitute rela-

tively pliable regions susceptible to repeated re-

gional gene rearrangments. Indications of multiple

rearrangements in the icm/dot region have indeed

been described (Ko et al., 2002a,b).

We sequenced 18 icm/dot genes and 4 other

genes in 18 dierent strains of L. pneumophila.

Comparative sequence analysis reveals a widespectrum of variability among the icm/dot genes,

some as conservative as mip and houskeeping

genes, others more variable than dotA. Protein

functional motif search along with the distribution

of variable/conservative regions along the gene

sequence gave additional information on the lo-

cation of functionally important domains in some

icm/dot gene products. We did not observe clear-cut associations of any particular gene variations

with either known serogroups or virulence phe-

notypes. Phylogenetic analysis indicated that dif-

ferent L. pneumophila strains displayed distinct

acquisition histories for some subsets of the icm/

dot genes, consistent with an evolutionary scenario

for the L. pneumophila species encompassing generearrangements as well as repeated horizontal gene

transfer events.

2. Materials and methods

2.1. Bacterial strains

The Legionella species and L. pneumophila

strains used in this study are enumerated in

Table 1.

2.2. Hybridization

Specic primers were designed to amplify all or

a portion of each gene in the Philadelphia 1 strainof L. pneumophila. In general, PCR was carried

out using 150200 ng DNA, 1.5mM MgCl2, 1reaction buer, 0.2 lM each dNTP, 10 pmol eachprimer, and 2U Taq polymerase (Invitrogen) in

50 ll reaction volumes with the following PCRprole (5min at 95 C; 35 cycles of 95 C, 30 s;

Leg 27 L. steigerwaltii SC-18-C9

Leg 28 L. parisiensis PF-209C-C2

tel, w

6 in 1

I. Morozova et al. / Plasmid 51 (2004) 127147 129Table 1

Legionella strains

L. pneumophila

Leg # Serogroup Isolate

Leg 1 1 Bellingham 1

Leg 2 11

Leg 3 1 Philadelphia 1

Leg 4 2 Togus 1

Leg 5 3 Bloomington 2

Leg 6 4 Los Angeles 1

Leg 7 5 Dallas

Leg 8 6 Chicago 2

Leg 9 7 Chicago 8

Leg 10 8 Concord 3

Leg 11 9 IN-23-G1-C2

Leg 30 13 82A31053

Leg 31 1 Knoxville 1

Leg 32 10 Leiden 1

Leg 33 11 797-PA-H

Leg 34 12 570-CO-H

Leg 35 1 Amsterdam B-1

Leg 36 1 Amsterdam B-2

Sources of strains: Leg 35 and 36, provided by Dr. R. van Ke

Netherlands ower show. Leg 35 was identied in 28 and Leg 3Dr. B. Fields at the CDC.Leg 29 L. rubrilucens WA-270A-C2

ere derived from the Legionnaires disease outbreak at the 1999out of 29 patients. The rest of the strains were obtained fromOther Legionella species

Leg # Legionella species Isolate

Leg 12 L. dumoi NY-23

Leg 13 L. longbeachae 1 LB-4

Leg 14 L. longbeachae 2 Tucker 1

Leg 15 L. gormanii LS-13

Leg 16 L. micdadei TATLOCK

Leg 17 L. wadsworthii 81-716

Leg 18 L. oakridgensis Oak Ridge-10

Leg 19 L. feeleii 1 WO-44C-C3

Leg 20 L. feeleii 2 691-WI-H

Leg 21 L. sainthelensis Mt.St.Helens-4

Leg 22 L. jordanis BL-54D

Leg 23 L. spiritensis Mt.St.Helens-9

Leg 25 L. jamestowniensis JA-26-G1-E2

Leg 26 L. cherrii ORW

lasmi52 C, 30 s; 72 C, 30 s; 7min at 72 C). The PCRproduct was radiolabeled using random primer

labeling kits RTS RadPrime (Life Technologies) or

Redi-Prime 2 (Amersham) with [a-32P]dATP ac-cording to the manufacturers instructions. EcoRI-digested Southern blots of 15 species of Legionella

other than pneumophila (17 strains in all), and 18

strains of L. pneumophila were probed with each

gene-specic amplimer under standard conditions(overnight hybridization at 65 C in 0.5MNaHPO4, pH 7.2, 7% sodium dodecyl sulfate

(SDS), 1% bovine serum albumin, 1mM ethy-

lenediamine tetraacetic acid, 125 lg/ml shearedsingle-stranded salmon sperm DNA), and then

washed to a relatively low stringency (75mM

NaCl/7.5mM Na3citrate/0.1% SDS, 65 C). In afew cases, hybridization temperature was reduced(45 C) and washing eliminated (just a single300mM NaCl/30mM Na3citrate/0.1%SDS room

temperature rinse).

2.3. PCR and sequencing

The same primer pairs were used to attempt to

amplify genes from various Legionella strains andspecies. When amplication failed, at least one

additional attempt was made with alternative pri-

mer pairs; moreover, in some cases it was neces-

sary to adjust the annealing temperatures for

individual strains or genes. After PCR, oligonu-

cleotides were dephosphorylated and primers de-

graded with shrimp alkaline phosphatase and

exonuclease I, respectively (incubation at 37 C,90min; enzymatic denaturation at 72 C, 15min).The same primers were then used for bidirectional

sequencing. With genes longer than about 500

bases, additional internal oligonucleotides were

designed for priming sequencing reactions. Most

sequencing reactions were done with ABI big dye

terminator kits or Amersham 377 energy transfer

kits, according to the manufacturers instructions,and following isopropanol precipitation, the se-

quencing products were separated on ABI 377 gel

systems (PerkinElmer). Individual sequence reads

were assembled into contigs using the Phrap

assembler (Green P., http://bozeman.mbt.wash-

ington.edu/) or SeqMan (Lasergene System,

130 I. Morozova et al. / PDNASTAR, Madison, WI). The quality of eachbase in each sequence was checked both auto-matically and manually. In a few cases where there

was uncertain base calling even after repeated se-

quencing attempts, these positions were eliminated

from the analyses.

The sequences obtained in this study have been

submitted to GenBank (National Center for

Biotechnology Information, Bethesda, MD). Ac-

cession numbers and the sequences themselves areavailable at http://genome3.cpmc.columbia.edu/~

legion/comp_proj.htm.

2.4. Additional gene sequences

Homologs of the icm/dot genes from C. burnetii

[sequence data were provided by The Institute for

Genome Research (TIGR) under an academic li-cense agreement] and Legionella longbeachae

(GenBank Accession No. gi18693262) were used

as outgroups in L. pneumophila gene analysis.

2.5. Sequence alignment and analysis

The ClustalW (Thompson et al., 1994) program

was selected for aligning nucleotide or translatedamino acid sequences. BioEdit version 5.0.6 (TA

Hall, http://www.mbio.ncsu.edu/BioEdit/bioedit.

html) and GeneDoc programs (H.B. Nicholas Jr,

http://www.psc.edu/biomed/genedoc/) were used to

manipulate the alignments and to build the protein

hydrophilicity proles. The number of nonsynony-

mous (leading to amino acid substitutions) (Kn) and

synonymous (silent) (Ks) nucleotide substitutionswere calculated using the MEGA 2.1 package

(Kumar et al., 2001, http://www.megasoftware .net/).

Both Kn and Ks were calculated per corresponding

site to avoid the inuence of overall gene composi-

tion. Four physico-chemical properties (volume,

polarity, charge, and hydrophobicity) were used to

characterize the results of amino acid substitutions

in comparisons of translated homologous se-quences (Bogardt et al., 1980; Kawashima and

Kanehisa, 2000). Corresponding dG values were

obtained usingMiyatasmatrix (Miyata et al., 1979)andwere calculated per one amino acid substitution

so that they would not depend on the rates of nu-

cleotide substitutions per se. Protein secondary

d 51 (2004) 127147structure predictionwas done using SSpro (Pollastri

1990) and PARACEL BLASTER machine were

used for the searches against the TIGR databases

Philadelphia 1 strain of L. pneumophila. The left

side of the autoradiogram shown in Fig. 1 depicts

some typical results. A table (Supplementary Table

SI) compiling the results for every gene and strain

is available online at http://genome3.cpmc.colum-

bia.edu/~legion/comp_project/comp_proj.htm. Inaddition to 16S rRNA genes, positive signals were

obtained in most species for housekeeping (e.g.,

asd) genes; in contrast, only some icm/dot genes

were detected in the non-pneumophila species (see

right side of autoradiogram in Fig. 1), chiey in

L. longbeachae (Leg14), and to a lesser extent in

Legionella dumo, Legionella wadsworthii, Le-

Fig. 1. Gene distribution in dierent legionellae as scored by

hybridization. Comparison of hybridization results using Phil-

adelphia 1 PCR amplied probes in L. pneumophila and other

Legionella species for 16S rRNA, aspartate b-semialdehydedehydrogenase (asd), one of the lvh and three of the icm genes.

From left to right: strains Leg 717. Presence of multiple bands

for 16S rRNA likely due to multiple copies of the rDNA in the

bacterial species (there are at least three partial or complete loci

in the Philadelphia 1 strain of L. pneumophila based on the

genomic sequence). Variation in banding patterns for other

genes could be due to presence or absence of paralogs, or to

EcoRI restriction site polymorphisms within or near the gene,

in the dierent organisms. In the case of the lvhB4 and other lvh/

lvr genes, variable patterns could reect the fact that they can be

located on a plasmid, as supported by recent data from our

laboratory (not shown).

lasmifor completed bacterial genomes (http://www.tigr.org) and the NCBI nonredundant databases

(http://www.ncbi.nlm.nih.gov).

2.7. Domain search

The SMART server (http://smart.embl-heidel-

berg.de (Letunic et al., 2002)) and PFAM database

(Bateman et al., 2002) were used to search for pro-tein functional domains and coiled-coil structures.

2.8. Phylogenetic analyses

Tree reconstruction and visualization were ac-

complished using the MEGA 2.1 package (Kumar

et al., 2001). The Li distance approach (Li et al.,

1985) was used for building the distance matrix.The neighbor-joining (NJ) tree-building algorithm

(Saitou and Nei, 1987), which builds a branching

tree diagram from the distance matrix by succes-

sively clustering pairs together, was used for phy-

logenetic inference. Condence levels of inferred

relationships were estimated following 1000 boot-

strap iterations. To address uncertainties of tree

branching, the split decomposition method of theprogram SplitsTree (Huson, 1998) was utilized.

Unlike most tree building methods, which force

data into a tree-like phylogeny, this method por-

trays the data in a mesh-like graph allowing con-

icting phylogenetic information to be visualized,

estimated, and compared.

3. Results

3.1. Gene composition of Legionella species

Low stringency hybridization to EcoRI-di-

gested Legionella DNA was carried out using la-et al., 2002), APSSP2 (Raghava, 2000), and PHDprograms (Rost, 1996).

2.6. Homology search

The generic BLAST program (Altschul et al.,

I. Morozova et al. / Pbeled amplied regions of selected genes from thed 51 (2004) 127147 131gionella gormanii, Legionella micdadei, Legionella

feeleii, and Legionella sainthelensis. Intermediateresults were obtained for the lvh/lvr genes (lvrA-E,

lvhB2-11, D4). For the most part, they generated

strong hybridization signals in L. dumo, L.

longbeachae (Tucker 1), L. wadsworthii, L. oak-

ridgensis, and L. cherrii, but weak or no signals in

the remaining species tested. These genes have

previously been shown not to be essential for

growth of L. pneumophila in macrophages (Segalet al., 1999). As an alternative to hybridization,

primer pairs from Philadelphia 1 were used in an

attempt to amplify genes directly from the other

strains and species. The results were usually con-

sistent with those obtained using hybridization;

with few exceptions, if negative results were ob-

imply that dierent strain virulence phenotypes arenot accounted for by simple presence or absence of

these orthologs. Therefore, to determine if more

subtle genetic features were involved, we carried

out a comparative sequence analysis on all these

genes in the dierent L. pneumophila strains as well

as the L. longbeachae icm/dot genes available from

GenBank. (Although we were able to amplify a

few of the icm/dot genes in the non-pneumophilaspecies, the comparative sequencing described be-

low was restricted to the pneumophila strains.)

3.2. Level of interstrain and interspecies variation in

L. pneumophila

gene

(96%

d ave

r dat

ubmi

r dot

ophil

132 I. Morozova et al. / Plasmid 51 (2004) 127147tained using one approach, negative results were

also obtained with the alternative procedure (seesupplementary Table SII at the above URL for all

the PCR results). Still, it is important to realize

that under the stringency conditions we utilized for

hybridization, we would not expect to identify

genes with less than 70% identity at the nucleic

acid level; similarly, at least 90% conservation of

primer sequence would be required for consistently

successful PCR amplication. Thus, an unob-served signal may be due either to true absence of a

gene, or perhaps more likely, substantial variation

in the genes sequence compared to that of Phila-delphia 1.

Among the L. pneumophila strains, high signal

strength was obtained for nearly every gene

(housekeeping, icm/dot, and lvr/lvh). This would

Table 2

Variations among Legionella genes

16S rRNAa Non-icm

DNA

Within L. pneumophila 99.2% 89100%

Between Legionella spp. 9199% (96%) 6999%

With Coxiellad 85.5%

The data in the above table represent averages or ranges (ana Based on our data and that of Adeleke et al. (1996).b Based on information available for 8 non-icm/dot genes, ou

et al. (1998), and Avison and Simm (2002).c Based on our data plus gene sequences for L. longbeachae s

(2002b) have shown a wider range of nucleic acid homology fo

comparisons the L. pneumophila subsp. fraseri and subsp. pneum

d TIGR data compared with the Philadelphia 1 strain of L. pneumThere are now about 48 known Legionella

species (Perez-Luz et al., 2002) and about 15L. pneumophila serogroups, comprising approxi-

mately 70 known serogroups in the genus overall.

Despite detailed analyses, there are complications

in some of the assignments (see review by Benson

and Fields, 1998). Appreciating that taxonomic

positioning cannot always accurately reect evo-

lutionary distance (Rosello-Mora and Amann,

2001), Table 2 summarizes icm and non-icm se-quence diversity based on our data for dierent

strains of L. pneumophila, and in a lesser number

of cases, other Legionellae, as well as published

gene sequence data. The dierences between Le-

gionella species are within or close to the standard

boundaries of speciation (for review, see Rosello-

Mora and Amann, 2001): 95% homology for 16S

sb icm/dot genesc

Protein DNA Protein

) 97.9% 9698% (97%) 94100% (98%)

7599% 6279% (70%) 5891% (74%)

3966% 2363%

rages) of the percent homology for dierent genes.

a plus that of Ratcli et al. (1997), Doyle et al. (1998), Ratcli

tted to GenBank by Rogers et al. (2002) (AF288617). Ko et al.

A within L. pneumophila (78100%), when they include in the

a.ophila.

rRNA and 70% for other genes. As can be seenfrom the table, icm/dot genes have a higher level of

inter-strain diversity (6279% homology, with a

mean value of 70%), than non-icm/dot genes,

though as of today, only L. pneumophila vs

L. longbeachae comparisons for several icm genes

are available.

There is a considerable range of variability for

the dierent icm genes among the L. pneumophilastrains examined both at the nucleotide and pro-

tein levels (Table 3). Some genes have a very low

percentage of variable positions, and even silent

substitutions are rare, while others, such as icmX,

have many polymorphic sites. There were no ma-jor insertions or deletions in the sequenced genes,

though there were a few 1 or 2 amino acid inser-

tions and deletions (e.g., in icmG in Leg7 and

Leg31; icmX in several strains).

The number of synonymous (Ks) and nonsyn-

onymous (Kn) nucleotide substitutions was deter-

mined per corresponding site, and the mean of all

pairwise strain comparisons was calculated foreach gene. The icmX,W, V, and dotA genes, which

are all members of icm/dot region I (the small icm

locus), are quite variable, showing consistently

higher Ks and Kn values (with the exception of Kn

Table 3

Sequence variations in icm/dot and non-icm genes

Gene Number

of strains

sequenced

Gene

length in

Leg 3

% nished Number of

polymorphic sites

Mean pairwise value per

site

Kn/Ksratio

dG per

one aa

changenucl aa Kn Ks

icmF 15 2922 100 278 50 0.005 0.075 0.067 0.923

icmB 16 3030 100 276 16 0.002 0.107 0.019 0.615

icmJ 17 627 99 46 5 0.002 0.105 0.019 1.167

icmD 14 399 99 39 4 0.002 0.092 0.022 0.566

icmC 18 582 100 52 10 0.006 0.111 0.054 0.688

icmG 18 807 99 94 27 0.012 0.131 0.092 1.063

icmK 18 1083 98 141 30 0.008 0.169 0.047 0.373

icmL 18 639 100 57 3 0.001 0.087 0.011 0.633

icmM 17 285 100 20 6 0.008 0.063 0.127 1.433

icmN 15 570 88 59 7 0.004 0.07 0.057 0.120

icmP 14 1131 90 95 12 0.002 0.077 0.026 0.034

icmQ 16 576 98 34 3 0.002 0.079 0.025 0.135

icmR 17 363 100 40 10 0.01 0.083 0.120 1.148

icmS 17 345 100 32 3 0.003 0.164 0.018 1.299

icmT 17 261 100 19 2 0.002 0.097 0.021 0.850

Mean for

the locus

0.005 0.101 0.048 0.736

e sub

one

rogen

I. Morozova et al. / Plasmid 51 (2004) 127147 133icmV 17 456 100 56

icmW 17 456 100 34

icmX 18 1404 99 298

dotA 5 3189 100 557Mean for

the locus

tphA 17 1257 99 159

asd 35 1020 99 29

rpp 17 690 100 74

RNAseH 11 573 100 22

mip 17 699 100 54

Both nonsynonymous (Kn) and synonymous (Ks) nucleotid

inuence of gene composition and dG values are calculated per

remainder. Abbreviations: asdaspartate b-semialdehyde dehydinammatory peptide.*Data from Bumbaugh et al., 2002.19 0.024 0.147 0.163 0.927

3 0.002 0.119 0.017 0.998

78 0.036 0.307 0.117 1.131

139 0.042 0.352 0.118

0.026 0.231 0.104 1.019

37 0.008 0.097 0.082 0.987

3 0.001 0.031 0.032 0.374

11 0.006 0.147 0.040 1.084

7 0.006 0.046 0.130 1.769

4 0.002 0.070 0.025

stitutions are calculated per corresponding site to avoid the

amino acid change. Numbers in bold vary the most from the

ase, rppagellar L-ring protein precursor, mipmacrophage

in the case of icmW), compared to most of thegenes from icm/dot region II. The icmX,V and

dotA genes have Kn values approximately 10 times

higher than most of the rest of the icm/dot genes.

The ratio of nonsynonymous to synonymous

nucleotide substitutions is usually taken as an in-

dicator of the functional and structural restrictions

on gene variability and is independent of the time

of gene diversication. The icm/dot genes show awide distribution in their Kn/Ks ratios, with, for

example, icmV having a ratio nearly 15 times

higher than that of icmL. The highly conserved

genes (icmL, W, S, B, J, T, D, P, and Q) have

lower Kn/Ks ratios than even the very conservative

housekeeping gene encoding aspartate b-semial-dehyde dehydrogenase (asd), which shows as much

as 62% homology even with its relatively distantVibrio cholerae ortholog. In contrast, the most

134 I. Morozova et al. / Plasmivariable genes (icmV, M, R, and X) have Kn/Ksratios close to or even higher than dotA, which

is considered a relatively variable gene (Bumbaugh

et al., 2002).

Not all amino acid substitutions in the geneswith

low Kn/Ks ratios are conservative, as assessed by

changes in amino acid physico-chemical properties,and there are cases of genes with relatively conser-

vative amino acid substitutions that nonetheless

have a high level of gene variability as judged byKn/

Ks ratios (Fig 2). For example, the IcmJ, S sand W

Fig. 2. Comparison of Kn/Ks and dG values for icm/dot and

non-icm/dot genes. Kn/Ks ratios and dG values for the icm/dot

genes shown in order from highest (icmV) to lowest (icmL) Kn/Ks values. Dashed lines correspond to locus II mean values.protein products, despite displaying relatively lowKn/Ks values, have amino acid substitutions that

result in drastic changes in their properties; on the

other hand, three genes (icmN, P, and Q) have

close to locus II average (0.05)Kn/Ks ratios, but their

encoded proteins have extremely low dG values,

indicating that only substitutions in amino acids

with similar physico-chemical properties have been

permitted. Since nucleotide substitutions may exerttheir inuence on the function of the nal protein

product at any of several levels (e.g., DNA, mRNA

or protein), Kn/Ks ratios reect general restrictions

on gene and protein variability. On the other hand,

dG values reect variation purely in protein

structural and functional features, indicating some

restrictions on the amino acid substitutions at

the level of the nal functioning product. In thissense, icmN, P, and Q may be considered the most

conservative of the icm/dot genes.

There is no obvious correlation between the

predicted cell localization of the protein products

of these genes and their variability levels. While

IcmN is thought to be an outer membrane protein

and not necessary for macrophage killing, IcmK is

an indispensable periplasmic or outer membrane,IcmP is an inner membrane, and IcmQ is a soluble

cytoplasmic protein required for pore formation

(Andrews et al., 1998; Coers et al., 2000; Dumenil

and Isberg, 2001; Segal and Shuman, 1998a;

Watarai et al., 2001).

Overall, the levels of sequence variation found

among the non-icm genes in L. pneumophila strains

(last group of genes in Table 3) and most of theicm genes from locus II were comparable to the

level of diversity in, for example, Salmonella ent-

erica housekeeping genes reported by Boyd et al.

(1997) (where the mean nonsynonymous to syn-

onymous nucleotide substitution ratio was 0.032).

The level of polymorphism among icm genes from

locus I (second group in Table 3) and some locus II

members exceeds signicantly that for both Le-gionella and Salmonella housekeeping genes and

most of the genes from icm/dot locus II, and cor-

responds to the variability level for the spaM and

spaN genes of the S. enterica inv-spa pathogen

invasion complex (Boyd et al., 1997).

The order of the icm/dot genes was apparently

d 51 (2004) 127147the same in all 18 strains we examined, as assessed

by our ability to amplify these genes using primersfrom the expected surrounding genes.

3.3. Paralogs of icm/dot genes in Philadelphia strain

of L. pneumophila

It is not unusual to nd distant homologs

among the genes of a single organism. These may

represent members of a gene family that carry outrelated but not identical functions, or they may no

longer have any functional properties in common.

Among the icm/dot genes, four partial homologs

(paralogs) for the 30 part of icmL (134 aa), one forits 50 portion (79 aa), and one for icmC wereidentied in a search of the now essentially com-

plete Philadelphia 1 genome (http://genome3.

180 aa. IcmC and IcmC1 have 40% identity over171 aa.

3.4. Further analysis of individual icm/dot genes

Multiple alignments of the icm/dot genes in all

the L. pneumophila strains under study permitted

more detailed sequence analyses. The sequence

variation patterns at both the nucleotide andamino acid levels, and dG and hydrophilicity

proles along the length of each ORF were de-

termined, as well as potential structural and

functional motifs. In Fig. 3, the distribution of

nucleotide and amino acid substitutions along the

nucleotide and corresponding amino acid se-

quences are compared for all the sequenced icm

s in t

ar ha

I. Morozova et al. / Plasmid 51 (2004) 127147 135cpmc.columbia.edu/~legion/). The icmL paralogsare located in dierent regions of the genome, and

the icmC1 paralog is separated from the locus II

icmC gene by 23 kbps. In each case, the paralogs

are surrounded by genomic housekeeping genes.

The average protein sequence homology between

IcmL and its 30 paralogs is relatively low but clear:31% identity and 52% similarity over an approxi-

mately 120 aa (amino acid) stretch. For compari-son, the L. pneumophila IcmL has 91% amino acid

identity with L. longbeachae IcmL over a 220 aa

stretch; 39% identity to C. burnetii IcmL over 200

aa; and 2530% identity to traM genes (Klebsiella

oxytoca, Pseudomonas syringae, Escherichia coli,

and Salmonella typhimurium plasmids) over 160

Fig. 3. Distribution of nucleotide and amino acid substitution

(bottom halves of each bar) and amino acid substitution (upper bis indicated with a vertical hatchmark.genes in L. pneumophila strains. Apparently, inmany cases, nonsynonymous substitutions (lead-

ing to amino acid changes in encoded proteins) are

distributed unevenly along the sequence. The gene

regions with low or no nonsynonymous substitu-

tions and close to average number of synonymous

substitutions are of special interest since the ob-

served conservatism cannot be explained merely by

too little evolutionary time for the compared se-quences to diverge. These regions, conservative at

the protein level, especially those preserved also in

distant homologous proteins, may correspond to

important protein domains, so where possible,

comparisons were made with distant homologs in

other bacteria in conjunction with the functional

he icm genes among L. pneumophila strains. Every nucleotide

lves) along the icm sequences from all the L.pneumophila strains

motifs predictions. A more detailed description ofsome of the icm genes (icmP, G, N, and K) follows.

3.5. IcmP

IcmP is believed to be an inner membrane

protein, possibly involved in DNA transfer, and

absolutely indispensable for macrophage killing(Segal and Shuman, 1998a). The gene product is

predicted to have a signal peptide (aa 135), trans-

membrane regions (aa 1739 and 92114) and a

trbA domain (aa 204372). trbA is one of the genes

found within the transfer region of IncI1 plasmids

such as R64, and is absolutely required for conjugal

transfer of these plasmids (Furuya and Komano,

1996). Although distant homologs of icmP arefound in Coxiella, Pseudomonas, and Salmonella,

they display a low overall level of sequence simi-

larity (1835% identity at the protein level); only in

the region of the trbA domain slightly increased

homology is found. Nonetheless, all the homologs

have very comparable hydrophilicity proles over

their entire lengths (Fig. 4). Since the gene has not

been allowed to accumulate signicant variableamino acid positions, it is likely to share a closely

related function in these fairly diverse genera.

Taking the 15 L. pneumophila strains as a

group, both synonymous and nonsynonymous

substitutions are distributed evenly along the icmP

Fig. 4. Hydrophilicity proles of IcmP and distant homologs.

RedL. pneumophila IcmP; blueCoxiella IcmP homolog;

greenPseudomonas sp. PyR19 plasmid conjugal-transfer re-

lated sequence SAT (gi 2642198); brownSalmonella typhimu-

rium R64 plasmid trbA gene (gi 20521502).

Fig. 5. Hopp and Woods hydrophilicity proles for IcmG and

its homologs. BlueL.pneumophila IcmG; redTraP of plas-

mid R64 gi 4903119; greenC. burnetii IcmG homolog.

quen

cum m

RE d

romy

136 I. Morozova et al. / Plasmid 51 (2004) 127147Fig. 6. Alignment of t-SNARE domains in assorted proteins. Se

japonicum Blr2548 protein (BAC47813); Clostridium acetobutyli

aeruginosa probable chemotaxis transducer (AE004706)t-SNA

minal end (D21267); SNAP25 N-terminal end (D21267); SacchaThe conservative amino acids are highlighted.ces (from top to bottom): L. pneumophila IcmG; Bradrhizobium

ethyl-accepting chemotaxis protein (AE007559); Pseudomonas

omains predicted by SMART system; human SNAP25 C-ter-

ces cerevisiae SEC9p proteinputative t-SNARE (NP_011523).

Fig. 9. Hydrophilicity prole of IcmK and distant homologs.

RedL. pneumophila; blueL. longbeachae; greenC. burnetii;

brownShigella TraN; blackKlebsiella TraN.

lasmisequence, but the gene appears to be very conser-vative, both at the nucleotide and amino acid

levels, with the lowest dG value of all the icm and

housekeeping proteins sequenced, especially in the

trbA region.

3.6. IcmG

IcmG has also been predicted to be an innermembrane protein; mutation of this gene leads to a

partial reduction in the bacterias ability to killmacrophages (Segal et al., 1998). When Legionella

pneumophila strains are compared, IcmG shows

elevated variability, both at the nucleotide and

protein levels. Variable positions are almost evenly

distributed along the sequence, except in the vi-

cinity of the C- and N-termini that lack evensynonymous substitutions.

Fig. 5 shows hydrophilicity prole comparisons

for icmG inLegionella and two distant homologs,C.

burnetii IcmG and plasmid TraP. Despite relatively

low sequence homology among the three genes (less

than 20% at the protein level), their predicted sec-

ondary structures (not shown) and hydrophilicity

proles display signicant similarity. Preservationof the protein structure in some cases may be more

important for a proteins function than the aminoacid sequence itself, and probably because of this,

structure-based methods of searching for distant

homologs are more ecient than sequence-based

approaches (Pawlowski et al., 2001; Sauder et al.,

2000). Examples of related bacterial proteins with

very low sequence identity but nearly identicalstructures are not uncommon (Bauer et al., 2001;

Ginalski et al., 2000; Girardeau et al., 2000).

For the IcmCTraQ (not shown) and IcmG

TraP comparisons, the protein similarity at these

higher structural levels is indeed stronger than at

the sequence level. Thus, despite sequence dis-

crepancies, the major function of these distant

homologs may remain intact. Local dissimilaritiesof the protein proles, as in the case of IcmG

TraP at positions 165185 (Fig. 5), require addi-

tional analysis. The Legionella and Coxiella IcmG

proteins, unlike their TraP homolog, are predicted

to have a t-SNARE domain precisely in this region

(aa 142210 in the Legionella IcmG protein; aa 95

I. Morozova et al. / P194 in C. burnetii homolog, which correspond topositions 153221 in aligned sequences in Fig. 5)and this similarity extends beyond the coiled-coil

structural features predicted for all three homologs

in this area (positions 123179) (Segal and Shu-

man, 1998b). [Weimbs et al. (1997, 1998) even

screen out coiled-coil features when performing

t-SNARE domain searches.] Proteins with t-

SNARE domains play important roles in mem-

brane fusion in eukaryotes (Weber et al., 1998).While the t-SNARE domains are highly diverse,

they usually possess a central glutamine (Q) resi-

due and preserve the overall domain structure

(Gotte and von Mollard, 1998; Weimbs et al.,

1998). There are only a few bacterial proteins

known to have similarity to the t-SNARE domain

(SMART Accession No. SM0397); most of these

are bacterial sensor and chemotaxis integralmembrane proteins. Several examples of these are

aligned with IcmG in Fig. 6. It will be interesting

to see if the t-SNARE domain is conserved in non-

pneumophila Legionella species with icm/dot loci. If

it is required for IcmG function during infection,

this feature may dierentiate the global function

of the Legionella icm/dot system from that of its

homologs in other organisms.

3.7. IcmN

IcmN is a putative outer membrane lipoprotein,

containing a signal peptide, and is dispensable for

macrophage killing (Segal et al., 1998). The se-

quence is well conserved, especially at the protein

levelamino acid substitutions among L. pneumo-phila strains occur only in the N-terminal half of the

protein, and the alternative amino acids always

have very similar physico-chemical properties (Figs.

2 and 3). An alignment of L. pneumophila and L.

longbeachae sequences also reveals that the C-ter-

minal half (after aa 90) is more conserved than the

N-terminal portion (Fig. 7). Starting at aa 83, the

IcmN protein shows weak homology to the OmpAdomain (Pfam F00691), which is found in bacterial

porin-like integral-membrane proteins and lipo-

proteins, most of which, like IcmN, have a con-

served OmpA domain within the C-terminal half

and a variable N-terminal portion. Some members

of this protein group have antigenic determinants,

d 51 (2004) 127147 137but IcmN does not display obvious hypervariable

. Dot

IcmN

ane pr

lasmiFig. 7. Alignment of IcmN gene product with distant homologs

(top to bottom with NCBI accession numbers): L. pneumophila

hypothetical protein (NP_249524); E. coli putative outer membr

138 I. Morozova et al. / Pregions. The alignment with several distant homo-

logs reveals two extremely conserved motifs:

QGVD at aa 147 and RVEIT at the C-terminus

(boxed in Fig. 7).

3.8. IcmK

The IcmK product is putatively a periplasmic orouter membrane protein, and possesses a secretion

signal peptide (Andrews et al., 1998); the protein is

needed for pore formation (Kirby et al., 1998),

indispensable for macrophage killing, but not

necessary for conjugation (Andrews et al., 1998;

Segal and Shuman, 1998a). It is homologous to the

plasmid traN gene product. According to the Pfam

database, the TraN domain starts at position 62 ofboth the protein alignment (Fig. 8) and the hy-

drophilicity prole for L. pneumophila icmK and

its distant homologs (Fig. 9); the alignment shown

before that point is uncertain owing to very low

homology.

As seen in the alignment, the homology level

between the orthologs is quite low with

lasmiI. Morozova et al. / Phydrophobic portion among distant homologs

suggests that this region is a functionally impor-

tant domain.

3.9. Phylogenetic relationships between strains

based on icm gene sequence

The dot/icm genes were presumably introduced

into the Legionella genomes from a plasmid

(Komano et al., 2000; Segal and Shuman, 1998a),

Fig. 8. Alignment of IcmK and TraN gene products. Dots represen

Sequences from top to bottom: L. pneumophila, L. longbeachae (AF2

ColIb-P9 TraN (BAA75158) (has only 1 aa dierence with Salmonell

oxytoca plasmid pACM1 primase (AF139719).d 51 (2004) 127147 139possibly prior to their separating into two loci. It is

unknown, though, if this was a one-time event or

the region(s) were lost and re-introduced repeat-edly during Legionella evolution. Often when gene

transfer occurs from a distant organism with dif-

ferent nucleotide content, the transferred region is

evident due to its dierent GC content compared

to the rest of the genome. In the case of Legion-

ellas icm/dot loci, their GC content is equivalent tothe genome average (38%). Moreover, the regions

t identical amino acids and dashes are gaps in the alignment.

88617), and C. burnetti IcmK; E. coli (Shigella sonnei) plasmid

a typhimurium IncI1 plasmid R64 TraN, BAB91663); Klebsiella

15 am

lasmiare distinct from those of their homologs in Cox-

iella and the R64 plasmid where most icm/dot gene

homologs are around 44 and 50% GC, respec-

tively. It is possible that the transfer occurred froma dierent plasmid with similar GC content to that

of Legionella. Based on the dierences between

phylogenetic trees built for mip and dotA (Bum-

baugh et al., 2002) and dotA and rpoB genes (Ko

et al., 2002a,b), it has been suggested that repeated

Fig. 10. icmK variability proles. A window size of

140 I. Morozova et al. / Pevents of genetic exchange or loss and acquisition

led to the current complex composition of these

loci.To determine if the rates of molecular evolution

of icm genes are disparate in dierent L. pneu-

mophila strains, a comparison of icm genes from

all available strains was undertaken, using their

C. burnetii orthologs as outgroups (Sexton and

Vogel, 2002). The distances in synonymous and

nonsynonymous substitutions per corresponding

site were analyzed separately, as was done byWhittam and Bumbaugh (2002). All analyzed

genes from all the L. pneumophila strains showed

approximately equal relative substitution rates

(data not shown). It is probable, though, that

minor dierences were missed, using such distant

homologs from Coxiella. In the future, when more

of the closer homologs, e.g., icm/dot genes from

other Legionella species, are available, it should bepossible to obtain a ner resolution.A detailed phylogenetic analysis was carried

out. Phylogenetic trees were built for 18 icm genes,

3 housekeeping genes, and the icmB/tphA inter-

genic region as well as combined trees built foricm locus subregions (i.e., concatenated icm genes

from extensive portions of the two loci or the en-

tire loci). The presented trees were built by two

methods: NJ, with 1000 bootstrap iterations to

estimate condence level for the tree topology, and

ino acids or codons was used. See text for details.

d 51 (2004) 127147the split decomposition method which displays

branching alternatives in a single representation.

While trees were built for each gene and severalicm/dot subregions, only some representative ex-

amples are included in Fig. 11.

Based on the combined phylogenetic trees, the

strains consistently group into seven subsets: [Leg

5, 1, 9], [6, 11, 32], [{36, 10}, {30, 35}], [3, 4, 8, 34],

[2, 33], and [7, 31], though the separation between

groups {36, 10} and {30, 35} is less consistent (cf.

Figs. 11A and C). This clustering is almost iden-tical, with a few exceptions, for the icm genes of

the two loci, houskeeping genes and the icmB/tphA

intergenic region and is supported by high boot-

strap values on almost all of the trees. Exceptions

to this clustering were most frequently found with

the Leg6 strain, which, for 6 icm and 3 house-

keeping genes, merges with the (5, 1, 9) group (see

for example the tree for icmK in Fig. 11D). Itappears that in the case of trees built for genes of

lasmiI. Morozova et al. / Pthe small locus (icmV, W, and X) and those at one

end of the large icm locus (icmF, tphA, icmB, J, D,

C, and G), Leg 6 belongs to the (11, 32) group,

whereas based on trees built for many of the genes

at the other end of locus II (icmK, L, N, R, S, and

T), this strain falls into the (5, 1, 9) group. In onlya very few cases was the clustering violated by

other strains (e.g., Leg 30 and 35 are in separate

branches in icmV, W, X, F, and B individual gene

trees).

Despite the largely consistent strain clustering,

the relationship between clusters is not as clear,

that is, the groups as a whole can switch their

relative positions in dierent trees and sometimescannot be positioned unambiguously (for example,

see Fig. 11D). In many cases, these cluster re-

Fig. 11. Phylogenetic trees. The gene sets do not include dotA,B,C, i

Combined NJ tree for all icm genes. (B) Aligned NJ trees for the two i

the text. Left: locus II (all locus II icm genes except icmF). Right: locus

for all icm genes. This gure shows the strain clustering, emphasizing t

of the strains. (D) Split decomposition tree for IcmK, demonstrating th

alternative branching.d 51 (2004) 127147 141locations have low bootstrap values, making it

dicult to judge whether they correspond to ac-

tual gene transfer or to recombination events.

In all the trees Leg 7 and 31 constitute a sepa-

rate group, so distant from the remaining strain

clusters that it almost has the appearance of anoutgroup. But when strains 7 and 31 are consid-

ered independently, they seem to be almost as

distant from each other as from the remaining

strains (Fig. 11C). Thus, they probably do not

form an actual group, but are merely the two most

divergent strains of L. pneumophila examined. It

was previously shown that L. pneumophila strain

Dallas, serogroup 5, which corresponds to our Leg7 strain, belongs to L. pneumophila subspecies

fraseri (Brenner et al., 1988) and that the dotA and

cmO or icmE. Numbers at the nodes are bootstrap values. (A)

cm loci. Notable dierences in the tree topologies are detailed in

I (icmX,W, and V only). (C) Combined split decomposition tree

he divergence of Leg 7 and 31 from each other and from the rest

e complicated picture of group branching. Rectangles represent

investigators have shown that among nine strains

of L. pneumophila, eight from serogroup 1 in-

lasmicluding three commonly used in laboratory studies

(AA100, JR32, and Lp01), the presence or absence

of two loci involved in Type IV secretion (traI andlvh) and the rtxA locus, may correlate to some

extent with the strains pathogenicities (Samrak-andi et al., 2002). More specically, the lvh and

rtxA loci were found more commonly in strains

generally associated with disease, whereas the traI

locus was not. These authors also were able

to detect and discriminate these genes by hybridi-

zation in some non-pneumophila species. Morerecently, dissection of an expanded locus sur-

rounding a set of the so-called tra/trb genes, pre-

sumably involved in pilus assembly, distinct from

the traI locus of the AA100 strain, as well as from

the icm/dot and lvr/lvh loci, revealed it to be a likely

pathogenicity island, containing additional genesmip genes from this strain were most distant fromtheir homologs in other L. pneumophila strains

(Bumbaugh et al., 2002).

The observed strain clustering does not correlate

with serogroups. Thus, while both Leg 2 and 33

belong to serogroup 11 and also to one cluster, none

of the ve strains of serogroup 1 for which we have

sequences (Leg 1, 3, 31, 35, and 36), group together.

Trees built for the locus I icm genes vary themost from the locus II genes (compare the two

combined trees in Fig. 11B, left and right). The

initial trees were aligned by rotating branches

around internal nodes, while preserving the

branching pattern, to accentuate the dierences

between the two resulting topologies. Branches

corresponding to strains Leg 5, 35, 1, and 6 could

not be aligned.

4. Discussion

The icm/dot gene loci are present in each of the

L. pneumophila serogroups and strains we se-

quenced. Moreover, based on our ability to am-

plify and sequence across genes of interest usingprimers in expected surrounding adjacent genes, it

appears that gene order within these clusters is also

retained within the L. pneumophila strains. Other

142 I. Morozova et al. / Pfor putative virulence factors such as methioninesulfoxide reductases, as well as plasmid mobilityelements; while present in Philadelphia 1-derived

strains, it appears to be missing in part or in its

entirety from JR32 and several clinical isolates

(Brassinga et al., 2003). Interestingly, this locus

contains paralogs of the lvrA, B, and C genes of

the lvr/lvh Type IV secretion locus. Perhaps the

most intriguing nding was the presence of a 30 kb

unstable genetic element in strain Olda but not inPhiladelphia 1 strains, possibly phage derived, in-

volved in phase variation (Luneberg et al., 2001).

When integrated into the chromosome, the strain

is virulent, but when excised and replicating as a

high-copy plasmid, it resultes in a mutant pheno-

type with a modied lipopolysaccharide O-antigen

epitope associated with reduced virulence.

At this point, we have insucient evidence todetermine if the icm/dot genes are absent or present

in most other Legionella species, with the excep-

tion of L. longbeachae where good hybridization

signals were obtained for icm C, D, G, K, L,M, O,

P, and T; weak signals with J, Q, R, S, V, and X;

and no signal for icm B, E, and F. Six L. long-

beachae icm/dot genes from the center of locus II

have been submitted to GenBank by T. Rogers, S.List, R.M. Doyle, and M.W. Heuzenroeder. In

cases where we do not obtain positive hybridiza-

tion signals using L. pneumophila probes, it is

probable that the orthologs are too dissimilar

in their sequence, at least in the region between

where the primers were designed, to be detected by

even the reduced stringency hybridization or am-

plication used in this study. Their characteriza-tion thus awaits large-scale sequencing of other

species, or the use of degenerate oligonucleotide-

based PCR. Terry Alli et al. (2003) recently re-

ported the presence of the icm/dot loci in every

Legionella species they examined based on hy-

bridization, even under high stringency conditions,

using pooled regional probes. While we did get

weak signals with many icm/dot genes in non-pneumophila species similar to the ones they dis-

played in their paper, we are unable to explain the

several cases of disagreement, except that we used

single gene probes which might have been too

species-specic. Since we probed the same blots

subsequently with several other probes for 16S

d 51 (2004) 127147rRNA, housekeeping or lvh/lvr genes and obtained

lasmiexcellent signals, the absence of hybridization withthose icm/dot gene probes can not be due to the

quality of the DNA itself.

The two icm/dot clusters may have been subject

to substantial changes in the course of their intra-

species evolution. Given that the icm/dot loci are

present in all the L. pneumophila strains from the

15 dierent serogroups we examined and the fact

that these strains have a 100-fold range in theirability to replicate within macrophages (data not

shown), it might be expected that the strains dif-ferences in virulence depend on sequence varia-

tions within the genes, especially in functionally

important gene and protein regions, such as those

responsible for ecient transport of eector mol-

ecules. Of course, it is also possible that altered

regulation of these genes (when and where they areexpressed), or in the eector molecules themselves,

can contribute to the pathogenic phenotype. It is

worth noting that even though the entire icm gene

set is present in the Coxiella genome (Seshadri

et al., 2003), its lifestyle is very dierent from that

of Legionella. In particular, Coxiella does not seem

to depend on the disruption of phagosomelyso-

some fusion for its survival, which is considered tobe the main function of the icm/dot system in Le-

gionella. In the current study, we assessed the level

of diversity among genes of the dot/icm loci, fo-

cusing on the putative functional domains that are

preserved even in distant homologs.

The dot/icm genes display a wide range of

variability, some being more conservative than

an average houskeeping gene (icmP, Q, D, T, J,B, S, W, and L), while others are 510 times

more variable (icmM, R, V, X, and dotA), as

indicated by the ratio of nonsynonymous and

synonymous nucleotide substitutions. Low vari-

ability at the sequence level, though, does not

necessarily mean that all the observed amino

acid substitutions are conservative with regard to

their physico-chemical properties. For example,it appears that IcmT, J, S, and W proteins are

permitted rather dramatic amino acid substitu-

tions. In contrast, IcmN, P, and Q are extremely

conservative at this level, but not as much at the

sequence level. In general, genes from locus I

show higher diversity compared to locus II, both

I. Morozova et al. / Pat the gene and protein levels.A second category of intra-species variation ispositional, with some portions of the genes and

their products more dissimilar than others. For

instance, the IcmK and IcmV proteins have many

more amino acid substitutions in their N-terminal

than their C-terminal portions. Most variation at

the amino acid level is found at the ends of IcmP,

but centrally in IcmG. At the same time, the silent

nucleotide changes are often distributed evenlyalong the gene indicating that the preservation of

amino acid sequence in some regions is not simply

due to time of gene divergence, but rather to the

presence of important functional domainsespe-

cially when the sequence, or at least the protein

structure, is preserved in distant orthologs. It is

interesting in this regard that remote homology

detection by structural methods has helped pre-dict the function of many otherwise uncharacter-

ized proteins in several sequenced genomes

(Pawlowski et al., 1999, 2001; Rychlewski et al.,

1998).

For some icm/dot genes (icmP, G, N, and K) the

combination of relatively high regional sequence

conservatism and the presence of predicted do-

mains and sequence and/or structure preservationin distant homologs in the same areas serve as

indicators of the presence of a functional domain,

though they await experimental proof. Features

such as the t-SNARE-like domain in IcmG and its

Coxiella homolog, occur rarely enough in bacterial

genes as to make them noteworthy. If the

t-SNARE domain is functional in IcmG, it may

compete with the hosts membrane fusion SNAREsystem, potentially altering its normal vesicular

tracking pathways, and preventing phagosome

lysosome fusion, for the bacterias own ends. Thusthese ndings may provide the impetus for future

experimental studies to more directly determine

the function of these proteins.

Phylogenetic analysis for individual genes as

well as locus subregions largely reveal similarstrain groupings, as in Fig. 11C. However, some

branches either switch their positions on dierent

trees or cannot be unambiguously positioned.

Though it is tempting to speculate that these rep-

resent instances of lateral transfer within the locus,

it is not possible to determine this with any

d 51 (2004) 127147 143certainty.

lasmiNot only are the locus I genes more variablethan most of locus II, but interestingly, genes of

the smaller locus (icmW, V, X, and dotA) have

accumulated more silent nucleotide substitutions

per site (Ks values) than most of those from locus

II. If both loci were acquired, probably from a

plasmid, at the same time, this may mean that

locus I is evolving at a higher rate. Alternatively,

under the assumption that the evolutionary rateshave been the same and unchanged for both loci,

genes from the smaller locus must be older than

most of those in the large icm cluster. This, taken

with the fact that the most disparate branching

patterns are observed when either individual or

combined trees for icm/dot locus I vs locus II are

compared, leads to the assumption that the icm/

dot region has a rather complex history of geneacquisition and rearrangment events. In Coxiella

all the icm genes are located next to each other

whereas in L. pneumophila they are split into two

icm/dot loci that are located on opposite sides of

the circular genome (http://genome3.cpmc.colum-

bia.edu/~legion/index.html). This may serve as an

additional indication that two loci in Legionella

were acquired separately or rearranged after-wards.

So far, full icm/dot gene sets have only been

found in two relatively close species (Legionella

and Coxiella), and this system diers substantially

from the known Type IV systems. Nonetheless,

given the presence of limited but obvious homol-

ogy of most icm/dot genes from both loci and tra/

trb genes, it is possible to suggest that they mayhave derived from the same ancestor. This ances-

tor may be of plasmid origin or assembled from

various chromosomal components in ancestral

bacteria; in the latter case, these genes may sub-

sequently have been incorporated into a plasmid,

support for which would come from the fact that

many dierent bacteria possess tra-like genes (e.g.,

Type IV secretion systems).Other researchers have also pointed out that

the icm/dot region may have a complicated evo-

lutionary history in L. pneumophila. Bumbaugh

et al. (2002) compared dotA and mip (a 24 kDa

surface protein with peptidyl-prolyl-cis/trans

isomerase activity that may be involved in es-

144 I. Morozova et al. / Ptablishment of infections, but not intracellularsurvival (Cianciotto et al., 1990), in 17 clinicaland environmental isolates. Compared to mip,

DotA, a cytoplasmic membrane spanning protein,

was extremely and perhaps unexpectedly variable,

and the neighbor-joining trees produced for the

two genes were discordant at several branch

points with high bootstrap values. The authors

considered this an indication of lateral gene

transfer and recombination and relatively recentgene dispersal. Ko et al. (2002b) compared the

dotA and rpoB alleles in 79 Korean isolates of L.

pneumophila from six clonal populations. The

most parsimonious tree produced using rpoB

distinguished four closely related L. pneumophila

pneumophila subspecies and two closely related L.

pneumophila fraseri subspecies. In contrast, in the

case of dotA, one of the pneumophila subspeciesseemed more closely related to the fraseri sub-

species than to the other three pneumophila. Some

caution should be exercised, however, in that

these authors previously showed that the rpoB

trees, themselves, diered substantially from 16S

rRNA and mip trees, which was the basis for

distinguishing the six clonal populations (Ko

et al., 2002a). Our comparisons, taking intoconsideration nearly all the members of the icm

dot loci, may point out additional subpopula-

tions, especially for those genes showing sub-

stantial variation.

In the future, comparisons with icm and lvh

plasmid gene orthologs may be especially inter-

esting. Since the lvh/lvr locus is likely to have been

inherited as a plasmid unit, as we discovered dur-ing the sequencing of the Philadelphia 1 genome

(manuscript in preparation), with a substantially

higher GC content (43%) than the rest of the ge-

nome (Segal et al., 1999), we intend to compare its

history with that of the icm/dot islands, which have

only some of the classic features of pathogenicity

islands (apparent absence of essential genes, all-or-

none presence of the complete gene set), but notothers (GC content the same as the remainder of

the genome, separation into two subsets). The

separate tra/trb locus also appears to be a patho-

genicity island, the central core of which has an

elevated GC content (Brassinga et al., 2003), and is

thus another good candidate for such comparative

d 51 (2004) 127147sequence analysis.

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I. Morozova et al. / Plasmid 51 (2004) 127147 145Forssmann, W.-G., Rosch, P., Adermann, K., Sticht, H.,

2001. Structure determination of human and murine b-defensins reveals structural conservation in the absence of

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Salmonella enterica. J. Bacteriol. 179, 19851991, id: 0021-Acknowledgments

Strains Leg 1Leg 34 were kindly provided by

Dr. Barry Fields at the CDC; Leg 35 and Leg 36,

specimens from an outbreak at a Dutch owershow, were a generous gift from Dr. Ruud van

Ketel at the University of Amsterdam. We thank

Huitao Sheng for assistance in sequence submis-

sion and Dr. Pavel Morozov for helpful comments

throughout the course of this work. This work was

supported by NIH Grant U01 1 AI 44371 awarded

to J.J.R., and funds generously provided by the

Columbia Genome Center.

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Communicated by R. Novick

Comparative sequence analysis of the icm/dot genes in LegionellaIntroductionMaterials and methodsBacterial strainsHybridizationPCR and sequencingAdditional gene sequencesSequence alignment and analysisHomology searchDomain searchPhylogenetic analyses

ResultsGene composition of Legionella speciesLevel of interstrain and interspecies variation in L. pneumophilaParalogs of icm/dot genes in Philadelphia strain of L. pneumophilaFurther analysis of individual icm/dot genesIcmPIcmGIcmNIcmKPhylogenetic relationships between strains based on icm gene sequence

DiscussionAcknowledgementsReferences