Mosaic Structure of Pathogenicity Islands in Legionella pneumophila

Kwan Soo Ko,1 Hae Kyung Lee,2 Mi-Yeoun Park,2 Yoon-Hoh Kook1

1 Department of Microbiology and Cancer Research Institute, Institute of Endemic Diseases,

Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital,

Seoul 110-799, Republic of Korea2 Laboratory of Rickettsial and Zoonotic Disease, Department of Microbiology, Korean National Institute of Health,

Seoul 122-701, Republic of Korea

Received: 9 September 2002 / Accepted: 9 January 2003

Abstract. A gene complex, dot/icm, located in twoindependent chromosomal loci of L. pneumophila, thecausative agent of Legionnaires’ disease, is related tovirulence. To investigate the evolutionary pattern ofthese pathogenicity islands of L. pneumophila, por-tions of four genes in the dot/icm complex, namely,dotA, dotB, icmB, and icmT, were amplified, se-quenced, and phylogenetically analyzed, in additionto rpoB, which encodes an RNA polymerase b-subunit. The nucleotide sequences and phylogeneticanalyses of these five genes of 96 L. pneumophilastrains revealed that several subgroups of L. pneu-mophila proliferated clonally. However, incongruentgene tree topologies and the results of statisticaltesting (Templeton Willcoxon signed-ranked and in-congruence length differences tests) indicated that theevolutionary histories of these genes within thepathogenicity islands are not uniform, and that theyconstitute a mosaic structure. In addition, the non-uniform grouping of some reference strains suggeststhat intraspecific recombination might be still occur-ring in nature or in the laboratory.

Key words: dot/icm — Recombination — Legion-ella pneumophila — Mosaic structure — Molecularevolution

Introduction

The gram-negative respiratory pathogen Legionellapneumophila is a facultative intracellular bacteriumthat infects and grows within human alveolar mac-rophages and protozoan host cells (Winn 1999). Ithas been postulated that L. pneumophila forms poresin the macrophage membrane and enter a vacuole,thus avoiding fusion with lysosomes when it is takenup by macrophages (Swanson and Hammer 2000). Itthen replicates inside the alveolar macrophagescausing a severe type of pneumonia called Legion-naires’ disease (Vogel and Isberg 1999). That is tosay, the most important characteristic of its virulencemay be its ability to prevent phagosome-lysosomefusion (Vogel and Isberg 1999). The alteration ofendocytic trafficking in phagosomes by L. pneumo-phila is mediated through the actions of productsencoded by dot/icm (defect in organelle trafficking/intracellular multiplication) genes (Berger and Isberg1993; Purcell and Shuman 1998; Setal et al. 1998;Vogel et al. 1998).Twenty-four dot/icm genes are located in two un-

linked 22-kb regions on the L. pneumophila chromo-some (Segal et al. 1998; Vogel et al. 1998).Chromosomal region I contains the genes icmV,icmW, and icmX, and dotA, dotB, dotC, and dotD,and chromosomal region II the genes icmT, -S, -R, -Q, -P, -N, -M, -L, -K, -E, -G, -C, -D, -J, and -B, andtphA and icmF (Segal et al. 1998; Vogel et al. 1998;Vogel and Isberg 1999). The Dot/Icm complex is

J Mol Evol (2003) 57:63–72DOI: 10.1007/s00239-002-2452-8

Correspondence to: Yoon-Hoh Kook; email: yhkook@plaza.snu.

ac.kr

thought to constitute a type IV-like secretion system,which is capable of injecting effector protein into thehost cell, that allows L. pneumophila to evade theendocytic pathways by modulating the activity of thehost factors involved in vesicle trafficking (Nagai etal. 2002; Zink et al. 2002). Moreover, L. pneumophilamutants defective in the Dot/Icm transporter systemcannot replicate intracellularly (Vogel et al. 1998;Andrews et al. 1998; Roy et al. 1998; Wiater et al.1998; Mathews and Roy 2000), which implies that thedot/icm complex is indispensable for the intracellularsurvival of L. pneumophila, and that its mode of ac-tion is evolutionarily conserved (Hilbi et al. 2001).It has been also reported that most of the Dot/Icm

proteins share significant amino acid sequence simi-larity with those of plasmid-encoded conjugationsystems, such as R64 (Segal and Shuman 1999; Ko-mano et al. 2000). Thus, it has been suggested that theR64 transfer of L. pneumophila and L. pneumophiladot/icm systems have evolved from a common an-cestral genetic system (Komano et al. 2000). In ad-dition, the dotA gene has been detected in otherLegionella species, such as L. micdadei, L. longbe-achae, L. bozemanii, and L. gratiana (Nagai et al.2002). In addition, homologues of icmT, icmS, andicmK have been found in Coxiella burnetii (Segal andShuman 1999), which is the causative agent of Q-fever and which is closely related evolutionarily toLegionella (Weisburg et al. 1989). These relationshipssuggest that the dot/icm complex was transferredfrom a plasmid into an unknown common ancestorof Legionella and Coxiella, and has since evolved.Thus, it is possible that the pathogenicity islands of

the L. pneumophila, dot/icm complex, have evolvednot only by plasmid incorporation into its chromo-some, but also by the intraspecific recombination ofcomplex after this plasmid incorporation. To verifythis we analyzed the genetic structure of four genes(dotA, dotB, icmB, and icmT) in the dot/icm complexof L. pneumophila to investigate their evolutionarypatterns. These four genes were selected on the basisof their importance in terms of their virulence anddisparity from one another. Along with these genes,

we also determined the sequence of a portion of thehousekeeping gene, rpoB. This gene encodes the b-subunit of DNA-dependent RNA polymerase (Se-verinov et al. 1996), which has been suggested to be analternative tool for determining the phylogeny of andfor identifying several bacteria (Mollet et al. 1997;Kim et al. 1999; Ko et al. 2002). It was interesting tonote that no concordance was found among thephylogenetic relationships inferred from the five genesequences, suggesting the complex molecular evolu-tion of the pathogenicity islands in L. pneumophila.

Materials and Methods

Bacterial Strains

Ninety-six strains of L. pneumophila were used for this analysis,

including 17 reference strains. Reference strains in all serogroups

(SGs) of L. pneumophila (SGs 1 to 15) were included in this study

except SG 9, because of no amplification of dotB, icmB, and icmT.

Four reference strains, ATCC 33152 (Philadelphia-1, type strain),

ATCC 33153 (Knoxville-1), ATCC 43109 (OLDA), and SF9, be-

longed to SG 1. Of the 79 culture isolates, 76 strains were isolated

from air-conditioner cooling water, and the three remaining strains

(KP1, KP2, and KP3) were obtained from the lung tissue of

pneumonia patients. These had been previously identified by se-

rologic and biochemical testing. For rpoB, dotA, and icmB, all 96

strains were included in the nucleotide sequencing and analyses,

but 37 strains, including 17 reference strains, were selected from

each L. pneumophila subgroup (Ko et al. 2002) and analyzed for

dotB and icmT.

Nucleotide Sequencing of Gene Fragments

The nucleotide sequences of the internal fragment of dotA, dotB,

icmB, and icmT were determined along with rpoB. The primers

used for this amplification and sequencing are shown in Table 1.

For PCR, 20 pmol of each primer were added to a PCR mixture

tube (AccuPower PCR PreMix; Bioneer, Daejeon, Korea), which

contained 1 unit of Taq DNA polymerase, and each deoxynucle-

oside triphosphate at a concentration of 250 lM, 10 mM Tris-HCl

(pH 8.3), 40 mM KCl, 1.5 mM MgCl2, and gel loading dye. The

bacterial culture suspension (1 ll) was amplified directly by PCR.The final volume was adjusted to 20 ll with distilled water, and thereaction mixture was subjected to 30 cycles of amplification. Each

cycle consisted of: 30 s at 95�C for denaturation, 30 s at 50–55�C

Table 1. Primers used for the amplification of each gene fragment and sequencing

Gene Lengtha Primers

rpoB 300 bp RL1 5¢-GAT GAT ATC GAT CAY CTD GG-3¢RL2 5¢-TTC VGG CGT TTC AAT NGG AC-3¢

dotA 360 bp dotAF 5¢-TTG ATT TGG TGA AAC TCA ATG G-3¢dotAR 5¢-CAA TCA AAA TCC TGG TGC TTC-3¢

dotB 346 bp dotBF 5¢-AAG ACG AAA GCC AAG ATT A-3¢dotBR 5¢-TGC GTC CCA AGT CAT TA-3¢

icmB 358 bp icmBF 5¢-GGC TGT TCC GGA AAT AAG AG-3¢icmBR 5¢-AGG GCA CAT GTG AAG GAA GAA-3¢

icmT 264 bp icmTF 5¢-AGG ATG ATT TTT TGA GG-3¢icmTR 5¢-TAT CTC GCT CCA TTT ATC T-3¢

a Length of sequenced fragment.

64

for annealing, and 30 s at 72�C for extension, and this was followedby a final extension at 72�C for 5 min (model 9700 Thermocycler;Perkin-Elmer Cetus). PCR products were detected on 1.5% agarose

gels stained with ethidium bromide and purified using a QIAEX II

gel extraction kit (Qiagen, Hilden, Germany) for sequencing. The

sequences of the purified PCR products were determined directly

with forward and reverse primers using an ABI377 automated se-

quencer and a BigDye Terminator Cycle Sequencing kit (Perkin-

Elmer Applied Biosystems, Warrington, United Kingdom). For the

sequencing reaction, 30 ng of purified PCR products, 2.5 pmol of

primer, and 4 ll of BigDye Terminator RR mix (Perkin-Elmer

Applied Biosystems; part number 4303153) were mixed and ad-

justed to a final volume of 10 ll with distilled water. The reactionwas run with 5% (vol/vol) dimethyl sulfoxide for 30 cycles of: 15 s

at 95�C, 5 s at 50�C, and 4 min at 60�C.

Phylogenetic Analysis

The five individual gene data sets of the 17 reference strains were

compared statistically for incongruence using the nonparametric

Templeton Wilcoxon signed-rank (WS-R) test (O’Donnell et al.

2001) and the incongruence length differences (ILD) test (or the

partition homogeneity test) (Farris et al. 1994; Cunningham 1997),

both of which are implemented in the PAUP* package (Swofford

1999). The maximum-likelihood (ML) method was also used to

determine the extent of congruence among the gene trees (Holmes

et al. 1999; Feil et al. 2001). For each gene, differences in log

likelihood (D-lnL) were calculated between the ML tree for a geneand the ML trees based on other genes. These differences in log

likelihood were compared with those of 200 randomly generated

trees for each gene. If the ML trees of each gene are congruent,

then all gene trees should have smaller differences in log likelihood

than those of the random trees (Holmes et al. 1999). This analysis

was also performed using the PAUP* program (Swofford 1999).

The phylogenetic tree of each gene fragment sequence was

constructed using the neighbor-joining method in PAUP*, with the

ML distance option of the HKY85 substitution model and no

among-site rate variation. All trees were rooted using the midpoint-

rooting option. Branch supporting values were evaluated by per-

forming 1000 bootstrap replications.

Nucleotide Sequence Accession Numbers

The nucleotide sequences determined in this study were submitted

to the GenBank database. The accession numbers of the reference

strains are AF367748, AF527122–AF527172, and AY036018–

AY036052.

Results

Sequence Diversity

Sequences of rpoB, dotA, and icmB were obtainedfrom 96 L. pneumophila strains and those of dotB and

icmT from 37 strains. The unambiguously determinednucleotide sequences in this study ranged from 264 bp(icmT) to 360 bp (dotA) (Table 1). Sequences wereedited and aligned using the EditSeq and MegAlignprograms (Windows version 3.12e; DNASTAR,Madison, WI). No insertions or deletions whatsoeverwere observed in any regions sequenced during thisstudy. At the nucleotide level, the maximum diver-gences of rpoB, dotA, dotB, icmB, and icmT were12.7%, 21.9%, 9.6%, 9.0%, and 5.2%, respectively.The deduced amino acid sequences were comparedwith previously published sequences (Segal et al.1998; Vogel et al. 1998). No amino acid substitutionwas found in RpoB. Polymorphisms were found atone site in DotB, six in IcmB, and five in IcmT, mostof which were shown by two subspecies of L. pneu-mophila (data not shown). On the other hand, ex-tensive substitutions, 48 out of 120 deduced aminoacids, were present in DotA.

Incongruence of Sequence Data Sets

Both the Templeton WS-R test and the ILD testshowed that the five individual gene data sets of the17 reference strains should not be combined witheach other. The Templeton WS-R test indicated thatall data sets were significantly incongruent (p <0.05). All the results of the ILD tests using 1000 re-plicates for 10 combined data sets also indicated lowcombinability (p < 0.01), i.e., incongruence (Cun-ningham 1997). The results of ML analysis of con-gruence between gene trees are presented in Table 2.The differences between the log likelihoods of trees(D-lnL) generated from the different genes fell insidethe 99th percentile of the random tree topologies intwo cases, the icmT tree in dotA and the icmB tree inicmT. Thus, all results showed that rpoB, dotA, dotB,icmB, and icmT of L. pneumophila might have beensubjected to different evolutionary pathways.

Subgroups of L. pneumophila Strains

Gene trees inferred from the five gene fragments areshown in Figs. 1–5. Previously, the population ofL. pneumophila strains was classified into six sub-groups (P-I to -IV for L. pneumophila subsp. pneu-

Table 2. Maximum-likelihood test of incongruence between genes

Gene

D-lnL of MLtree

D-lnL of ML trees fromother genes

99th percentile D-lnLin random trees

Genes outside 99th percentile

of random trees

rpoB 778.494 78.437–88.125 191.932 –

dotA 1291.084 486.992–799.685 620.121 icmT

icmB 767.652 113.072–196.813 241.185 –

dotB 760.399 113.596–160.354 241.107 –

icmT 513.483 66.896–120.010 84.713 icmB

65

mophila, and F-I and -II for L. pneumophila subsp.fraseri), according to the rpoB and dotA gene analysis(Ko et al. 2002). Six subgroups were also valid inother gene (icmB, dotB, and icmT) trees with the ex-ception of subgroup P-I in the icmB tree. Thoughsubgroup P-III of subsp. pneumophila was close to thetwo subgroups of subsp. fraseri in the dotA tree (Fig.2), two subspecies of L. pneumophila were not inter-mixed in the five gene trees indicating the geneticseparation of the two subspecies (Ko et al. 2002). Inthe icmB tree, strains of the P-I subgroup did notform a monophyletic cluster. Instead, subgroup P-IIwas inserted within the main clade of subgroup P-I(Fig. 3), and the icmB sequences of subgroup P-IIdiffered by only one or two nucleotides from those ofsubgroup P-I.

Interrelationship Between the L. pneumophilaSubgroups

In contrast to the general conservation of subgroup-ings within L. pneumophila, interrelationships be-tween the subgroups were not concordant (Figs. 1–5).No pair of gene trees showed the same tree topologyamong the subgroups. Though the two subgroups ofL. pneumophila subsp. fraseri (F-I and F-II) wereseparated, they were nevertheless closely related withthe rpoB, dotA, and dotB trees, and were not sepa-rated in the icmB and icmT trees. However, foursubgroups of L. pneumophila subsp. pneumophila didnot show consistent relationships, In the rpoB tree,which is believed to give reliable relationships (Ko etal. 2002), subgroups of L. pneumophila subsp. pneu-

Fig. 1. Neighbor-joining tree based on

the rpoB gene sequence. Thirty-eight

strains selected in dotB (Fig. 4) and in

icmT (Fig. 5) are represented as asterisks

at the right of the strain names. Desig-

nation of L. pneumophila subgroups, P-I

to P-IV and F-I to F-II from Ko et al.

(2002).

66

mophila formed a single cluster, which was distinctlyseparated from that of L. pneumophila subsp. fraseri(Fig. 1) and from the icmB and dotB trees.

Placements of Reference Strains ofL. pneumophila in Gene Trees

Table 3 shows the subgroups of the 17 referencestrains. Only the five reference strains of L. pneu-mophila subsp. pneumophila, ATCC 43109 (OLDA,SG 1), ATCC 33154 (SG 2), ATCC 33155 (SG 3),ATCC 33215 (SG 6), and ATCC 43290 (SG 12),and the two reference strains of L. pneumophilasubsp. fraseri, ATCC 33156 (SG 4) and ATCC33216 (SG 5), belonged to identical subgroups in allgene trees. The other reference strains clustered into

different subgroups in different gene trees. For ex-ample, the Philadelphia-1 (ATCC 33152, SG 1) typestrain of L. pneumophila belonged to P-I in the rpoBand dotA trees but to P-III in the icmB, dotB, andicmT trees. Morever, the dispositions of ATCC33153 (Knoxville-1, SG 1), ATCC 43283 (SG 10),and ATCC 35251 (SG 15) into subgroups differedin each gene tree (Table 3). In addition, SF9 (SG 1),ATCC 33823 (SG 7), ATCC 35096 (SG 8), ATCC43736 (SG 13), and ATCC 43703 (SG 14) did notbelong to any subgroup in some of the gene trees,though they merged into one of the subgroups inothers (Table 3), with the exception of ATCC 43130(SG 11), which was not included in any subgroup inthe five gene trees. All isolates consistently belongedto the same subgroups in the five gene trees without

Fig. 2. Neighbor-joining tree based on

the dotA gene sequence. Thirty-eight

strains selected in dotB (Fig. 4) and in

icmT (Fig. 5) are represented as asterisks

at the right of the strain names.

67

any exception, while several reference strains didnot.

Discussion

The integration of foreign DNA into the bacterialchromosome is an important aspect of the evolutionof genomes and the emergence of new pathogens(Hacker et al. 1997; Hacker and Kaper 2000). Genesencoding the proteins of the Type IV secretion systemwere introduced into bacterial genomes from plas-mid, because their coding genes have been identifiedon many self-transmissible plasmids (Christie andVogel 2000; Nagai and Roy 2001). The Dot/Icmsystem of L. pneumophila seems to be a protein

transporter of the type IV secretion system, as isfound in Brucella sp., Bordetella pertussis, Helico-bacter pylori, and Rickettsia prowazekii (Christie andVogel 2000). This study suggests that the dot/icmcomplex of L. pneumophila has evolved by the in-corporation of a plasmid into its chromosome and bycomplex intraspecific recombination after this incor-poration.The gene trees inferred in this study show that

genes within the dot/icm complex have experienceddifferent evolutionary routes. If genes within the dot/icm complex had been transmitted en bloc and onlyvertically from one generation to the next, the phy-logenetic relationships of each gene would be identi-cal (Holmes et al. 1999; Kalia et al. 2002). However,the phylogenetic relationships among the six sub-

Fig. 3. Neighbor-joining tree based on

the icmB gene sequence. Thirty-eight

strains selected in dotB (Fig. 4) and in

icmT (Fig. 5) are represented as asterisks

at the right of the strain names.

68

groups in the five gene trees were quite different (Figs.1–5). Incongruence tests, such as the Templeton WS-R, the ILD, and the ML tests, confirmed the differentevolutionary paths of the individual genes. Such in-congruent tree topologies and sequence data sets canbe explained by horizontal gene transfer (Feil andSpratt 2001; Feil et al. 2001).However, the preservation of clonality in each

subgroup, except subgroup P-I in the icmB tree, in-dicates that recombination among individuals ofL. pneumophila is not as free as in H. pylori (Suer-baum et al. 1998). The congruence of subgroupingsamong the isolates may reflect genetic barriers to geneflow between the different subgroups within a popu-lation, i.e., cryptic speciation (Maynard Smith et al.1993; van Belkum et al. 2001). Horizontal genetransfer in L. pneumophila may be sporadic and its

limited clonality can be explained by the periodicemergence of fitter genotypes, which would give fre-quent rise to clones (Levin 1981). For example, thecloseness of P-III, which is a subgroup of subsp.pneumophila, to L. pneumophila subsp. fraseri (F-Iand F-II) in the dotA tree (Fig. 2) might be a relic of apast horizontal gene transfer of dotA from L. pneu-mophila subsp. fraseri to the ancestor of subgroup P-III (Ko et al. 2002). After the integration of foreignDNA, including dotA, the dotA gene of each sub-group might have evolved independently by muta-tion, and not by recombination. Thus, the clonality ofeach subgroup may have been preserved.Strains of subgroup P-II were inserted into those

of subgroup P-I, and two clades of subgroup P-I didnot cluster with most isolates of subgroup P-I in theicmB tree (Fig. 3). This may indicate that horizontal

Fig. 4. Neighbor-joining tree based on the

dotB gene sequence.

Fig. 5. Neighbor-joining tree based on

the icmT gene sequence.

69

gene transfer of icmB from subgroup P-I to subgroupP-II has occurred recently. Due to the relatively re-cent intragenic recombination of icmB, subgroups P-Iand P-II are not separated clearly in a gene tree. Aftersuch a sporadic gene transfer, the icmB of subgroupP-II might have maintained segregated clonal prolif-eration from subgroup P-I by a single nonsynony-mous mutation (R268 fi K268). In addition, there isno evidence to suggest that the two distinct clades ofsubgroup P-I constitute other clonal complexes, be-cause these isolates showed only one or two nucleo-tide differences and did not form a distinct cluster inany other gene tree.On the other hand, intragenic recombination be-

tween certain subgroups of L. pneumophila subsp.pneumophila and subsp. fraseri seemed to have oc-curred long before or the mutation rate must havebeen high. Moreover, the intermediate positions ofsubgroup P-IV in the dotA tree (Fig. 2) and subgroupP-II in the icmT tree (Fig. 5), along with their pres-ervation of clonality may support this proposal.Subgroup P-IV in icmB (Fig. 3) and subgroup P-II indotB (Fig. 4) may also indicate the occurrence ofsporadic recombination events or high mutationrates.However, the inconsistency of subgroupings in

several reference strains suggests that the recombi-nation of genes within the dot/icm complex may be inprogress. Whether the intragenic recombination ofreference strains occurred naturally in the environ-ment is not clear. Recently, it was reported that Lp01and JR32, which originate from the Philadelphia-1strain (SG 1, type strain), and which show geneticand phenotypic differences (Samrakandi et al. 2002),have been used in many molecular pathogenesisstudies of L. pneumophila. Therefore, it was inferredthat such differences are due to the different passagehistories of strains. Coupled with such observations,it is feasible to suggest that such gene recombinationin L. pneumophila may have occurred in the labora-tory. It is important to note that such a recombina-tion would affect the phenotype, i.e., the virulenceand the susceptibility of a bacterium to antimicrobialagents (Feil and Spratt 2001; Samrakandi et al.2002).dotA showed more dissimilarity than the other

four genes both in nucleotide and deduced aminoacid sequences. This extensive diversity seems to haveoriginated from its nature. DotA is a kind of poly-topic membrane protein (Roy and Isberg 1997), andis secreted into culture supernatant by Dot/Icmtransporter (Nagai and Roy 2001). Thus, the diver-sity of amino acids via lateral gene transfer and/orpoint mutations may increase the fitness of L. pneu-mophila in certain environmental niches, such aswithin a particular biofilm community or species ofamoebae (Berger et al. 1994; Bumbaugh et al. 2002)T

able

3.Subgroupsthatreferencestrainsbelongedtoinfivegenetreesa

SG1b

SG1c

SG1d

SG1e

SG2

SG3

SG4f

SG5f

SG6

SG7

SG8

SG10

SG11

SG12

SG13

SG14

SG15f

rpoB

P–I

P–II

P–I

P–I

P–III

P–II

F–II

F–I

P–III

––

P–IV

–P–III

P–I

P–IV

F–I

dotA

P–I

P–II

–P–I

P–III

P–II

F–II

F–I

P–III

––

P–II

–P–III

–P–IV

F–II

icmB

P–III

P–IV

P–I

P–I

P–III

P–II

FF

P–III

––

P–IV

–P–III

P–I

P–IV

F

dotB

P–III

P–II

P–I

P–I

P–III

P–II

F–II

F–I

P–III

––

P–II

–P–III

––

F–II

icmT

P–III

P–II

P–I

P–I

P–III

P–II

FF

P–III

P–II

P–III

P–IV

–P–III

–P–IV

F

aBoxesindicateidenticalsubgroupingsinfivegenetrees.ForL.pneumophilasubsp.fraseri,dottedlineswereshown.

bATCC33152(Philadelphia–1).

cATCC33153(Knoxville–1).

dSF9.

eATCC43109(OLDA).

fStrainsthatbelongedtoL.pneumophilasubsp.fraseri.TheothersareL.pneumophilasubsp.pneumophila.

70

in terms of immune surveillance system evasion. Thehigh variation shown by dotA may also be related toadaptation and intracellular survival in different hostspecies, such as various species of amoeba, ciliates,and other protozoans (Swanson and Hammer 2000).However, nucleotide and deduced amino acid diver-gences of the other genes within the pathogenicityislands (icmB, dotB, and icmT) were found to besimilar to or lower than those of the housekeepinggene, rpoB. This indicates that though icmB, dotB,and icmT may have been exchanged by intraspecificrecombination, no selective pressure has diversifiedthem further.This study suggests that complex recombination

has occurred after the acquisition of blocks of path-ogenicity islands in L. pneumophila. Such intraspecificrecombination within pathogenicity islands has notbeen previously reported. In spite of a recent clonalproliferation and the presence of distinct subgroupsin L. pneumophila, it is evident that there have beenintraspecific recombinations and that the relation-ships between major subgroups of L. pneumophilashould be depicted as a network rather than a tree.In other words, the pathogenicity islands of L.pneumophila are mosaic-like in structure.

Acknowledgments. This work was supported by a grant of the

Korea Health 21 Research and Development Project, Ministry of

Health and Welfare, Republic of Korea (01-PJ10-PG6-01GM03-

0002), and in part by the BK21 project for Medicine, Dentistry,

and Pharmacy.

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