J Mol Evol (1999) 49:627–644© Springer-Verlag New York Inc. 1999

Phylogenetic Analysis of Pseudomonas syringae Pathovars Suggests the
Horizontal Gene Transfer of argK and the Evolutionary Stability of hrp
Gene Cluster
Hiroyuki Sawada,1 Fumihiko Suzuki,1 Izumi Matsuda,2 Naruya Saitou3
1

National Institute of Agro-Environmental Sciences, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8604, Japan
National Agriculture Research Center, Tsukuba, Ibaraki 305, Japan
3
National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
2

Received: 18 January 1999 / Accepted: 25 May 1999

Abstract. Pseudomonas syringae are differentiatedinto approximately 50 pathovars with different plant
pathogenicities and host specificities. To understand its
pathogenicity differentiation and the evolutionary
mechanisms of pathogenicity-related genes, phylogenetic analyses were conducted using 56 strains belonging
to 19 pathovars. gyrB and rpoD were adopted as theindex genes to determine the course of bacterial genome
evolution, and hrpL and hrpS were selected as the representatives of the pathogenicity-related genes located on
the genome (chromosome). Based on these data, NJ, MP,
and ML phylogenetic trees were constructed, and thus 3
trees for each gene and 12 gene trees in total were obtained, all of which showed three distinct monophyletic
groups: Groups 1, 2 and 3. The observation that the same
set of OTUs constitute each group in all four genes suggests that these genes had not experienced any intergrouphorizontal gene transfer within P. syringae but have beenstable on and evolved along with the P. syringae genome. These four index genes were then compared withanother pathogenicity-related gene, argK (the phaseolotoxin-resistant ornithine carbamoyltransferase gene,which exists within the argK–tox gene cluster). All 13
strains of pv. phaseolicola and pv. actinidiae used had
been confirmed to produce phaseolotoxin and to have
argK, whose sequences were completely identical, without a single synonymous substitution among the strains

Correspondence to: H. Sawada; e-mail: sawada@niaes.affrc.go.jp

used (Sawada et al. 1997a). On the other hand, argK
were not present on the genomes of the other 43 strainsused other than pv. actinidiae and pv. phaseolicola.
Thus, the productivity of phaseolotoxin and the possession of the argK gene were shown at only two points on
the phylogenetic tree: Group 1 (pv. actinidiae) andGroup 3 (pv. phaseolicola). A t test between these twopathovars for the synonymous distances of argK and thetandemly combined sequence of the four index genes
showed a high significance, suggesting that the argK
gene (or argK–tox gene cluster) experienced horizontal
gene transfer and expanded its distribution over two
pathovars after the pathovars had separated, thus showing a base substitution pattern extremely different fromthat of the noncluster region of the genome.
Key words: argK-tox gene cluster — hrp gene cluster— gyrB — rpoD — Molecular phylogeny — Pseudomonas syringae — Horizontal gene transfer

Introduction
Pseudomonas syringae is genetically diverse and is now
subclassified into approximately 50 pathovars according
to plant pathogenicity and host range (Bradbury 1986;
Dye et al. 1980; Rudolph 1995). However, such a pathovar system does not always conform with DNA homology or physiological and biochemical characteristics andmay be hindering the analyses of pathogenicity differen-628

tiation and diversity of P. syringae (Bradbury 1986; Rudolph 1995; Young et al. 1992).
To understand the pathogenicity differentiation of P.
syringae, it is necessary to study not only the phenotypic
expression of pathogenicity but also the genes that codefor the phenotype. In general, plant pathogenic bacteria
must have various factors that are related to host plant
affinity and directly involved in destroying plant tissues
to intrude into and infect plants and cause disease. Thus,
the number of pathogenicity-related genes should beenormous (Baker et al. 1997; Daniels et al. 1988; Gopalan and He 1996; Rich and Willis 1997; Rudolph1995). Moreover, there are data suggesting that dynamic
evolutionary mechanisms such as horizontal gene transfer and genome rearrangement are involved in the diversification of pathogenicity-related genes existing on theP. syringae genome (“genome” refers to “bacterial chromosome” in this paper) and have accelerated and complicated the pathogenicity differentiation (Hatziloukas
and Panopoulos 1992; Sawada et al. 1995a, 1996, 1997a, b;
Sawada, unpublished data). Therefore, to understand thecomplicated pathogenicity differentiation of P. syringae,
we should systematically analyze (1) what pathogenicity-related genes are present and functioning in eachstrain, (2) how each gene has mutated and differentiated,(3) whether the gene has experienced dynamic evolutionary changes such as horizontal gene transfer and genome
rearrangement, (4) what its origin and molecular mechanisms of its dynamic evolution are, and (5) what newgene combinations have appeared on the genome
through the dynamic evolution.
Based on this viewpoint, we started to investigate thediversity of P. syringae by comparing its genes. We first
selected the 16S–23S rRNA intergenic spacer sequencesin the rrn operon as the index and discovered that P.
syringae pathovars have diverse spacer sequences(Sawada et al. 1995a, 1996, 1997a). At the same time, we
also found that their 16S–23S spacers are not appropriate
as an index for phylogenetic analyses since the insertionand deletion of several to dozens nucleotides frequently
occur in the 16S–23S spacer (Sawada et al. 1995a, 1996,
1997a; Sawada, unpublished data). Thus, a proteincoding gene in which base substitution is a basis of mutation and horizontal gene transfer seldom takes placeshould be found and used as a new index.
The objectives of this study are to analyze (1) the
evolutionary course of the P. syringae genome, (2) when
and which pathogenicity-related genes arose or were introduced by horizontal gene transfer on each genome,and (3) how those genes mutated and differentiated, using the newly adopted index genes. The index genes
adopted here to determine the course of P. syringae genome evolution were gyrB (the DNA gyrase B subunitgene) (Huang 1996; Yamamoto and Harayama 1995;
Yamamoto and Harayama 1996) and rpoD (the primary
sigma factor gene, or the group 1 ␴70-type sigma factorgene) (Gruber and Bryant 1997; Lonetto et al. 1992),
which are indispensable single-copy genes on whichhorizontal gene transfer seldom occurs, and widely accepted indices for phylogenetic analyses. We used 56
strains belonging to 19 pathovars, including theirpathotype strains, and sequenced the two adopted index
genes to clarify the course of genome evolution. Then we
selected hrpL and hrpS as the representatives of thepathogenicity-related genes, which exist in all P. syringae pathovars and are believed to be involved in thebasic process of determining the pathogenicity and hostrange (Baker et al. 1997; Gopalan and He 1996), andcompared the course of genome evolution determinedfrom gyrB and rpoD genes with the data on hrpL and
hrpS genes. We also compared the data of these fourgenes with those of another pathogenicity-related gene,argK [the phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) gene, which exists within the argK–
tox gene cluster], whose sequence we analyzed previously (Sawada et al. 1995a, 1997a; Sawada, unpublisheddata).
The study revealed that these P. syringae pathovars
used differentiated into three monophyletic groups, theargK gene (argK–tox gene cluster) had been distributedin two pathovars through horizontal gene transfer, andthe hrp gene cluster (hrpL and hrpS) had always beenstable on the genome and never underwent horizontal
gene transfer between groups within P. syringae. Preliminary reports of this study have been presented orally
at the annual meetings of the Phytopathological Societyof Japan (Sawada et al. 1995a, 1996, 1997b).

Materials and Methods

Bacterial Strains
Fifty-six strains belonging to 19 P. syringae pathovars, including theirpathotype strains, were used (Table 1). The sources of the strains used
and their relevant characteristics are also shown in Table 1. Taxonomic
positions of P. syringae strains were confirmed by identifying theirphenotypic features according to standard methods (Hildebrand et al.
1988; Takikawa et al. 1989).

Gene Amplification and Nucleotide Sequencing
Template preparation, PCR amplification, and nucleotide sequencing
were performed according to the methods described previously(Sawada et al. 1995b, 1997a).
Oligonucleotide primers amplifying the target regions were
designed based on the published sequence: for gyrB (gyr-F,
5⬘-CGCCAGGGTTTTCCCAGTCACGACCMGGCGGYAAGTTCGATGACAAYTC-3⬘; and gyr-R, 5⬘-TTTCACACAGGAAACAGCTATGACTRATBKCAGTCARACCTTCRCGSGC-3⬘);
for rpoD (rpo-F, 5⬘-CGCCAGGGTTTTCCCAGTCACGACAAGGCGARATCGAAATCGCCAAGCG-3⬘; and rpo-R, 5⬘TTTCACACAGGAAACAGCTATGACGGAACWKGC-

AR1
AC30
AC30
AC30
AC30
AC30
AP1
BR1
CA1
ER1
GL1
GL1
JA1
LA1
LA2
LA2
MA1
MA2
MA2
MA2
MA2
MR1
MR2
MR6
MR6
MR6
MR6
MP1
MP2
MP3
MY1
PA1
PA1
PA1
PA1
PA1
PA1
PA1
PU4
PI1
PI2

pv. aceris
pv. actinidiae

pv. pisi

pv. myricae
pv. phaseolicola

pv. morsprunorum

pv. mori

pv. maculicola

pv. japonica
pv. lachrymans

pv. aptata
pv. broussonetiae
pv. castaneae
pv. eriobotryae
pv. glycinea

OTU namea
Species name of host plant (common name)Acer sp. (maple)
Actinidia chinensis (kiwi fruit)
Actinidia chinensis (kiwi fruit)
Actinidia chinensis (kiwi fruit)
Actinidia chinensis (kiwi fruit)
Actinidia arguta (tara vine)
Beta vulgaris var. saccharifera (sugarbeet)
Broussonetia kazinoki x B. papyrifera (paper mulberry)
Castanea crenata (chestnut)
Eriobotrya japonica (loquat)
Glycine max subsp. max (soybean)
Glycine max subsp. max (soybean)
Hordeum vulgare (barley)
Cucumis sativus (cucumber)
Cucumis sativus (cucumber)
Cucumis sativus (cucumber)
Brassica oleracea var. botrytis (cauliflower)
Brassica pekinensis
Brassica campestris (Chinese cabbage)
Brassica campestris (Chinese cabbage)
Brassica campestris (Chinese cabbage)
Morus alba (mulberry)
Morus alba (mulberry)
Morus alba (mulberry)
Morus alba (mulberry)
Morus alba (mulberry)
Morus alba (mulberry)
Prunus domestica (European plum)
Prunus mume (mume, Japanese apricot)
Prunus salicina (plums)
Myrica rubra (myrica)
Phaseolus vulgaris (kidney bean)
Phaseolus vulgaris (kidney bean)
Phaseolus vulgaris (kidney bean)
Phaseolus vulgaris (kidney bean)
Pueraria lobata (kudzu vine)
Pueraria lobata (kudzu vine)
Pueraria lobata (kudzu vine)
Pueraria lobata (kudzu vine)
Pisum sativum (pea)Pisum sativum (pea)Strain designationb

MAFF 302273PT
KW–11PT
MAFF 302091
MAFF 302092
MAFF 302145
MAFF 302133
MAFF 302253PT
KOZ 8101PT
C–3PT
MAFF 302259PT
MAFF 302260PT
MAFF 301683
MAFF 301072PT
MAFF 302278PT
MAFF 301315
MAFF 301321
MAFF 302264PT
MAFF 301174
H–1
H 3–6
H 3–9
MAFF 302279PT
MAFF 301020
S 6801
S 6806
S 7133–2 (Sm2)S 7233–1 (Sm4)MAFF 302280PT
MAFF 301436
W 8010MAFF 301464PT
MAFF 302282PT
MAFF 301673
MAFF 301022
MAFF 301616
TMR 327
MAFF 301766
KUZ 8
KUZ 1
MAFF 302269PT
MAFF 301208
USA
Shizuoka, Japan
Kanagawa, Japan
Kanagawa, Japan
Wakayama, Japan
Kanagawa, Japan
USA
Tottori, Japan
Shiga, Japan
USA
NZ
Ibaraki, Japan
Tochigi, JapanUSA
Gunma, Japan
Chiba, Japan
NZ
Kanagawa, Japan?c?
?
Hungary
Nagano, Japan
Gunma, JapanTokyo, Japan
Shimane, Japan
Shimane, Japan?
Shiga, Japan
Aomori, Japan
Okinawa, Japan
CanadaIbaraki, Japan
Hokkaido, Japan
Hokkaido, Japan
Shizuoka, Japan
Ibaraki, Japan
Yamagata, Japan
Ibaraki, Japan
NZ
Shizuoka, Japan

Geographic
originc

Strains of Pseudomonas syringae used and data bank accession numbers for DNA sequences determined in this study

Taxon (pathovar)

Table 1.

MAFF
Y. Takikawa
MAFF
MAFF
MAFF
MAFF
MAFF
M. SatoK. Takanashi
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
M. SatoM. SatoM. Sato
MAFF
MAFF
M. SatoM. SatoM. SatoM. Sato
MAFF
MAFF
K. Takanashi
MAFF
MAFF
MAFF
MAFF
MAFF
Y. Takikawa
MAFF
M. SatoM. Sato
MAFF
MAFF

Sourceb
AB016299
AB016303
AB016303
AB016303
AB016303
AB016303
AB016307
AB016311
AB016315
AB016319
AB016323
AB016323
AB016327
AB016331
AB016335
AB016335
AB016339
AB016343
AB016343
AB016343
AB016343
AB016347
AB016351
AB016355
AB016355
AB016355
AB016355
AB016359
AB016363
AB016367
AB016371
AB016375
AB016375
AB016375
AB016375
AB016375
AB016375
AB016375
AB016379
AB016383
AB016387

gyrB
AB016300
AB016304
AB016304
AB016304
AB016304
AB016304
AB016308
AB016312
AB016316
AB016320
AB016324
AB016324
AB016328
AB016332
AB016336
AB016336
AB016340
AB016344
AB016344
AB016344
AB016344
AB016348
AB016352
AB016356
AB016356
AB016356
AB016356
AB016360
AB016364
AB016368
AB016372
AB016376
AB016376
AB016376
AB016376
AB016376
AB016376
AB016376
AB016380
AB016384
AB016388

rpoD

AB016301
AB016305
AB016305
AB016305
AB016305
AB016305
AB016309
AB016313
AB016317
AB016321
AB016325
AB016325
AB016329
AB016333
AB016337
AB016337
AB016341
AB016345
AB016345
AB016345
AB016345
AB016349
AB016353
AB016357
AB016357
AB016357
AB016357
AB016361
AB016365
AB016369
AB016373
AB016377
AB016377
AB016377
AB016377
AB016377
AB016377
AB016377
AB016381
AB016385
AB016389

hrpL

DDBJ accession No.d

AB016302
AB016306
AB016306
AB016306
AB016306
AB016306
AB016310
AB016314
AB016318
AB016322
AB016326
AB016326
AB016330
AB016334
AB016338
AB016338
AB016342
AB016346
AB016346
AB016346
AB016346
AB016350
AB016354
AB016358
AB016358
AB016358
AB016358
AB016362
AB016366
AB016370
AB016374
AB016378
AB016378
AB016378
AB016378
AB016378
AB016378
AB016378
AB016382
AB016386
AB016390

hrpS

629

SY1
SY2
SY2
SY5
SY7
TB1
TB1
TB1
TH3
TH3
TH3
TH2
TH2
TH2
TO1

pv. syringae

Species name of host plant (common name)
Syringa vulgaris (common lilac)
Syringa vulgaris (common lilac)
Syringa vulgaris (common lilac)
Prunus persica (peach)
Citrus iyo (Iyo)
Nicotiana tabacum (tobacco)
Nicotiana tabacum (tobacco)
Nicotiana tabacum (tobacco)Camellia sinensis (tea)Camellia sinensis (tea)Camellia sinensis (tea)Camellia sinensis (tea)Camellia sinensis (tea)Camellia sinensis (tea)
Lycopersicon esculentum (tomato)

Strain designationb
MAFF 302155T
MAFF 301861
LOB 2–5
W 7835
T–1
MAFF 302270PT
MAFF 301612
PT–13
PT1PT
S2
Tea 632
K 9301
K 9302
1
MAFF 302272PT

If two strains had one nonidentical gene sequence, both strains were kept for further analyses andgiven the respective OTU names regardless of whether the other three gene sequences were identical.
If strains showed all four gene sequences to be identical, one strain was selected from one pathovar
as an OTU. In this way, we selected 31 strains of 56 as OTUs, gave each strain the OTU name, andused the OTU names in the figures, tables, and text.
b
MAFF—Microorganisms Section of the MAFF Gene Bank, National Institute of Agrobiological
Resources, Tsukuba, Ibaraki 305-8602, Japan; T. Miyoshi—Takanori Miyoshi, Ehime Fruit Tree
Experiment Station, Japan; Y. Nishi—Yatsuka Nishi, Kagoshima Tea Experiment Station, Japan; M.

apv. tomato

pv. theae

pv. tabaci

OTU namea

Continued

Taxon (pathovar)

Table 1.

MAFF
MAFF
Y. Takikawa
K. Takanashi
S. Shigeta
MAFF
MAFF
T. Miyoshi
Y. Takikawa
Y. Nishi
Y. Nishi
Y. Nishi
Y. Nishi
Y. Nishi
MAFF

Sourceb
AB016391
AB016395
AB016395
AB016399
AB016403
AB016407
AB016407
AB016407
AB016411
AB016411
AB016411
AB016415
AB016415
AB016415
AB016419

gyrB
AB016392
AB016396
AB016396
AB016400
AB016404
AB016408
AB016408
AB016408
AB016412
AB016412
AB016412
AB016416
AB016416
AB016416
AB016420

rpoD

AB016393
AB016397
AB016397
AB016401
AB016405
AB016409
AB016409
AB016409
AB016413
AB016413
AB016413
AB016417
AB016417
AB016417
AB016421

hrpL

DDBJ accession No.d

AB016394
AB016398
AB016398
AB016402
AB016406
AB016410
AB016410
AB016410
AB016414
AB016414
AB016414
AB016418
AB016418
AB016418
AB016422

hrpS

Sato—Mamoru Sato, National Institute of Sericultural and Entomological Science, Japan; S. Shigeta—
Susumu Shigeta, Yamaguchi Prefectural Nishiki Agricultural Extension Center, Japan; K. Takanashi—Kazuo Takanashi, Hokkou Chemical Co., Ltd., Japan; Y. Takikawa—Yuichi Takikawa, Facultyof Agriculture, Shizuoka University, Japan. PT, pathotype strain; T, type strain.
c?, information was not available.
d
All the listed sequences were determined in this study. The same sequences within each OTU are
represented by one accession number.

UK
Nagano, Japan
Nagano, Japan
Fukushima, Japan
Yamaguchi, Japan
Hungary
Akita, Japan?
Shizuoka, Japan
Shizuoka, Japan
Shizuoka, Japan
Kagoshima, Japan
Kagoshima, Japan
Kagoshima, JapanUK

Geographic
originc

630

631
GCAGGAAGTCGGCACG-3⬘); for hrpL (hrpL-F, 5⬘CGCCAGGGTTTTCCCAGTCACGACTTTTGGCTGGCAYGGTTATCGCTATA-3⬘; and hrpL-R, 5⬘-TTTCACACAGGAAACAGCTATGACTGTGGTTTTGCGTGCGAGTTGGTTCC-3⬘); andfor hrpS (hrpS-F, 5⬘-CGCCAGGGTTTTCCCAGTCACGACCTSCAGGCCAAGCTGCTGAGGGTGC-3⬘; and hrpS-R, 5⬘TTTCACACAGGAAACAGCTATGACTTGAGCTCRCGGATATTGCCGGGCC-3⬘).
Both the forward (F) and the reverse (R) primers contained thesequences of Cy5-labeled primers (ALFred M13-40 and ALFred M13
Reversal) for cycle sequencing reactions, which are indicated by underscores (the sequence of primer ALFred M13-40 was added to forward primers, and that of primer ALFred M13 Reversal to reverse
primers). Both strands were sequenced directly by cycle sequencing,using these Cy5-labeled primers.

support for a particular tree topology by summing up the estimatedlog-likelihoods of individual genes for that topology, and compared thetotal log-likelihoods for different topologies, using TotalML (Adachi
and Hasegawa 1996).

DNA Sequence Alignments

DNA Sequence Accession Numbers

Raw sequence data were analyzed and compiled using DNASIS-Mac
version 3.6 (Hitachi Software Engineering) and MacClade version 3.06
(Maddison and Maddison 1996) softwares, and sequence alignments
were facilitated using CLUSTAL W version 1.7 (Thompson et al.
1994). Several typical DNA sequences were selected from raw data andconverted into amino acid sequences, and multiple alignments among
these were inferred. These results and the published alignments data
were used as templates to conduct multiple alignments of all the DNA
sequences determined and to correct them manually. Functional informations on the gene products were also used to aid in the multiplealignments. The multiple alignments are available in machine-readableform. For information, e-mail sawada@niaes.affrc.go.jp. Positions
where gaps are present in any one of the aligned sequences were
excluded from the following analysis.

The DNA sequences which we determined in this study have been
deposited in the DDBJ/EMBL/GenBank international nucleotide sequence database under the accession numbers shown in Table 1. The
same sequences within each OTU are represented by one accession
number.

Phylogenetic Analysis
Phylogenetic trees were constructed using three tree-building methods:
the neighbor-joining (NJ) method (Saitou and Nei 1987), the maximum-parsimony (MP) method (Fitch 1977), and the maximumlikelihood (ML) method (Felsenstein 1981).
Evolutionary distances (number of base substitutions) were estimated using the one-parameter (Jukes and Cantor 1969) and twoparameter (Kimura 1980) methods, and these distances were used forconstructing NJ trees using MEGA version 1.0 (Kumar et al. 1993),
CLUSTAL W, and PHYLIP 3.572c (Felsenstein 1996). Nonsynonymous and synonymous substitutions per site were calculated by themethod of Nei and Gojobori (1986) using MEGA.
PAUP version 3.1.1 (Swofford 1993) was used for MP analysis,
and heuristic and branch-and-bound searches were used to ensure finding the most-parsimonious trees. When two or more parsimonious trees
were obtained, we constructed a strict consensus tree and a 50% majority-rule consensus tree.
In looking for the ML tree topology, the local rearrangement
searches of NucML and ProtML (contained in the program package
MOLPHY version 2.3) (Adachi and Hasegawa 1996) were carried out,
starting from the NJ tree topology and/or the topology obtained by thequick add OTUs search option as the initial trees. The HKY85 model(Hasegawa et al. 1985) was used for the base substitution process, andthe JTT-F model (“F” option of the JTT model) (Cao et al. 1994) for
the amino acid substitution process. When the heterogeneity in rate was
detected among the three codon positions of protein-coding genes, arealistic model distinguishing among rates at different codon positions,
namely, the rate-heterogeneous model (Hasegawa and Adachi 1996),
was adopted. When a single gene did not contain sufficient phylogenetic information to resolve the problem at hand, we collectively usedfour index genes (gyrB, rpoD, hrpL, and hrpS), evaluated the total

Reliability Test for Inferred Tree Topology
To evaluate the reliability of the inferred tree topology, the bootstrap
probability (Felsenstein 1985) was calculated for trees inferred by the
NJ and MP methods by repeating the bootstrap resampling procedure1000 or 10,000 times. For the ML method, the local bootstrap probability (LBP) was calculated using the RELL method (Adachi and
Hasegawa 1996; Hasegawa and Kishino 1994; Kishino et al. 1990)
with 1000 replications.

Results
Sequence Determination and Alignment
To confirm that the gyrB and rpoD genes, which wereselected as the indices for determining the evolutionary
course of the P. syringae genomes, are actually single
copies on bacterial genomes, we studied the databases offive types of eubacteria [Haemophilus influenzae, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp. (PCC 6803), Escherichia coli K-12] for which
the sequences of the entire genomes are determined andfound that there was only one copy identified or estimated to be gyrB or rpoD on the genome of each bacterium (data not shown). A Southern analysis also confirmed that these two genes are both single copies on theP. syringae genomes used in this study (Sawada, unpublished data). Moreover, the GC contents of these genes,
their GC contents at the third codon positions, and thefrequency of codon usage showed that both genes had
characteristics similar to those of other genes existing on
the P. syringae genome (Sawada, unpublished data).
Hence, both genes likely have never experienced horizontal gene transfer or gene duplication but had evolved
on and along with the P. syringae genome. Therefore, we
concluded that gyrB and rpoD are appropriate indices foranalyzing the evolutionary course of P. syringae genomes.
gyrB. We determined the partial sequences of gyrB
genes (612 bp) for 56 strains, which belong to 19 pathovars of P. syringae (Table 1). The sequence determinedcorresponds from position 1501 to position 2115 on E.
coli K12 numbering (accession number X04341). We

632

used 615 bp of the gyrB sequences of P. aeruginosa(Yamamoto, personal communication) and E. coli (accession number X04341), which correspond to the P.
syringae gyrB partial sequences determined in this study,
as outgroups (OTU names: PAE and ECO, respectively).
(Unless stated otherwise, sequence data are expressed inthis paper with the OTU names shown in Table 1.)
As a preliminary test, two sequences (BR1 and PAE)
were converted into amino acid sequences with DNASIS
and were aligned with ClustalW. Then, we conducted
multiple alignment for all the DNA sequences of gyrB
used in this study with ClustalW and corrected the resultsmanually based on the amino acid sequence alignment of
BR1 and PAE.
rpoD. We determined the rpoD partial sequences of
56 strains used in this study (Table 1). For most strains,
the sequence of 516 bp in the coding region was determined, but we obtained data on 522 bp for pv. eriobotryae (ER1) due to inserted nucleotides in this region. The
sequences determined correspond to positions 3362–
3874 of E. coli K-12 numbering (accession number
J01687) and cover most of the “nonconserved insertion”
located between region 1 and region 2 of RpoD (Lonetto
et al. 1992). Since this section is less conserved than theother regions of rpoD, we expected that it would besuitable for the index of intraspecific comparison. The
rpoD sequences of P. aeruginosa (accession number
D90118) and E. coli (J01687) of 519 and 513 bp, respectively, which correspond to the P. syringae data determined here, were used as outgroups (OTU names: PAE
and ECO, respectively).
As a preliminary test, five sequences (AC30, ER1,
BR1, PAE, and ECO) were converted into amino acidsequences and were multiple aligned. Then we conducted multiple alignment for all the DNA sequences of
rpoD used in this study, and corrected the results manually based on the amino acid sequence alignment obtained by the preliminary test and on that reported by
Tanaka and Takahashi (1991).
hrpL. We determined the sequence of the entire hrpL
coding region (555 bp) for 56 strains used. The sequenceof the entire algU coding region of P. aeruginosa (582bp; U49151) and the sequence of the entire rpoE coding
region of E. coli (576 bp; U37089) were used as outgroups. AlgU, RpoE, and HrpL are all alternative sigma
factors of the ECF subfamily that belongs to the group 3
␴70-type sigma factor (Lonetto et al. 1994). After multiple alignment of these DNA sequences, we corrected
the results manually based on the amino acid sequencealignments reported by Martin et al. (1994) and Wei andBeer (1995).
hrpS. We determined the partial sequence (240 bp) of
the hrpS coding region for 56 strains used. This 240-bp

region corresponds to positions 577–816 of the P. syringae pv. phaseolicola numbering (M28524) and constitutes a part of domain D of prokaryotic regulatory proteins that interact with ␴54-RNA polymerase holoenzyme(Drummond et al. 1986; Grimm and Panopoulos 1989).
We used 240 bp of P. aeruginosa fleR (L41213) and E.
coli tyrR (M12114), which correspond to the P. syringae
hrpS partial sequences determined here, as outgroups (allof these code for prokaryotic regulatory proteins).
Selection of OTUs. We compared the sequences of 56
strains and found strains that have identical sequences. If
two strains had one nonidentical gene sequence, both thestrains were kept for further analyses regardless ofwhether the other three gene sequences were identical. If
strains showed all four gene sequences being identical,
one strain was selected from one pathovar as an OTU. In
this way, we selected 31 strains of 56 as OTUs, gave
each strain the OTU name shown in Table 1, and usedthem for further analyses with two outgroups (ECO andPAE).
Since the object of this study, P. syringae, is one
species, the analyses were intraspecific comparison.
Moreover, evolutionary distances between any two
OTUs of P. syringae should be very small (Figs. 1 and
2). Thus, the saturation of base substitution shouldscarcely affect the analyses. In addition, we determined the base composition of each gene and confirmedthat the compositions did not much vary among P. syringae OTUs (data not shown). Therefore, discussions in
the following sections are based mainly on DNA sequence analyses and use amino acid sequences for reference.

Analyses of Each Gene by Three Phylogenetic Tree
Drawing Methods
gyrB. We conducted NJ bootstrap analysis of finallydetermined gyrB sequence alignments, each of 612 bp,
from which the insertion/deletion sites had been removed. The resulting NJ tree showed three large groupsof P. syringae (Fig. 1): Group 1, which contained 9
OTUs (TO1, LA1, MA1, MA2, SY7, MP1, TH2, TH3,AC30); Group 2, with 8 OTUs (AR1, SY1, SY2, SY5,
JA1, AP1, PI1, PI2); and Group 3, with 14 OTUs (GL1,
PA1, PU4, MR1, MR2, MR6, BR1, MP2, MP3, MY1,
TB1, ER1, CA1, LA2). These groups were formed withbootstrap probabilities of 100, 100, and 75.9%, respectively. The outgroups (ECO and PAE) formed a root
between Group 1 and the other two groups, and thus the
topology that shows the group branching order was(1,(2,3)) (Fig. 1, Table 2). The bootstrap probability for
determining the group branching order was relatively
high (94.5%).

633

Fig. 1. NJ tree expressing the evolution of the P. syringae gyrB gene,based on 612 bp of the gyrB gene sequence, from which sites that
include gaps in more than one sequence had been excluded, constructedusing the distance matrix derived from Kimura’s (1980) two-parameter
method (see text for details). The tree was rooted using gyrB sequencesof P. aeruginosa (Yamamoto, personal communication) and E. coli
(X04341) as the outgroups. Horizontal branch lengths are proportional
to the estimated number of nucleotide substitutions, and bootstrap probabilities (as percentages), as determined for 1000 resamplings, aregiven above or beside the internal branches. Abbreviations for strains(OTU names) are as listed in Table 1.

The topology of the ML tree also showed threegroups, Groups 1, 2, and 3 (data not shown). The OTUs
forming each group were identical to those of the NJ tree.
The branching order of these groups was also identical tothat of the NJ tree and was (1,(2,3)) (Table 2). However,the local bootstrap probability determining the branchingorder was lower (77.6%).
The MP tree also showed three groups (data notshown). The OTUs constituting each group and thebranching order of the three groups were identical to the
NJ and ML results, but the bootstrap probability determining the branching order was lower (65.3%) (Table 2).

Fig. 2. NJ tree expressing the evolution of the P. syringae rpoD gene,based on 510 bp of the rpoD gene sequence (see text and legend to Fig.
1 for details). The tree was rooted using rpoD sequences of P. aeruginosa (D90118) and E. coli (J01687) as the outgroups. Horizontal
branch lengths and bootstrap probabilities are as described in the legend to Fig. 1. Abbreviations for strains (OTU names) are as listed inTable 1.

rpoD. We analyzed rpoD sequence alignments eachof 510 bp, from which insertion/deletion sites had been
removed, using the NJ and MP methods. Like the gyrB
analyses, these analyses also showed three groups (Fig.
2). The OTUs constituting each group and the branchingorder of the three groups were identical to the gyrB results (Table 2). The bootstrap probabilities for the branching orders were both low, 69.8 and 73.0%, respectively.
The ML tree also showed three groups and OTU constitutions identical to those of the NJ and MP trees (datanot shown). However, the branching order of these threegroups, (2,(1,3)), was different (Table 2). The local bootstrap probability determining the branching order was
low, 72.8%.

634
Table 2.

Tree topology (group branching order) obtained by three phylogenetic tree drawing methods
Tree topology (group branching order)a

Sequence

Method

gyrB

NJ
ML
MPc
NJ
ML
MPc
NJ
ML
MPc
NJ
ML
MPc

rpoD

hrpL

hrpS

Tandemly combined sequenceof all 4 genes(3,(1,2))(2,(1,3))(1,(2,3))

Bootstrap probability (%)b

X
X
X
X

94.5
77.6
65.3
69.8
72.8
73.0
53.4
55.1
88.1
54.0
51.6
55.4

X
X
X
X
X
X
X
X

NJ
ML
MPc

X

86.9
67.0
60.9

X
X

a

Group 1 contained 9 OTUs (TO1, LA1, MA1, MA2, SY7, MP1, TH2,
TH3, AC30); Group 2 contained 8 OTUs (AR1, SY1, SY2, SY5, JA1,
AP1, PI1, PI2); and Group 3 contained 14 OTUs (GL1, PA1, PU4,
MR1, MR2, MR6, BR1, MP2, MP3, MY1, TB1, ER1, CA1, LA2) (seeFigs. 1–4).
b
The bootstrap probability that determines the group branching order.

For the ML method, the local bootstrap probability (LBP) was calculated using the RELL method (Hasegawa and Kishino 1994; Kishino etal. 1990) with 1000 replications.
cWhen two or more parsimonious trees were obtained, the topology ofa strict consensus tree was used.

hrpL. The NJ, ML, and MP trees determined from thehrpL data, each of 555 bp, from which gaps had been
eliminated, showed the same three groups as the above
analyses (data not shown). The OTUs constituting eachgroup were also identical to the above results. The
branching order of the groups was (1,(2,3)) in the NJ tree
but was (2,(1,3)) in the ML and MP trees (Table 2). The
bootstrap probabilities for the orders were all low, 53.4,
55.1, and 88.1%, respectively.

Table 3.

hrpS. All the NJ, ML, and MP trees showed threegroups as in the other three genes (data not shown). The
OTUs constituting each group were also identical to theother results. The branching order of the groups was(1,(2,3)) in the NJ and MP methods and (2,(1,3)) in the
ML method (Table 2). The bootstrap probabilities for theorders were all low, 54.0, 55.4, and 51.6%.

Confirmation of Monophyly of the Three Groups that
Appeared in the Gene Trees
All 12 phylogenetic trees (gene trees) described in theprevious section showed an identical grouping pattern of
31 P. syringae OTUs: all trees showed three groups(Groups 1, 2, and 3). We confirmed the monophyly ofeach group by checking each gene.
Confirmation by Bootstrap Probability. The bootstrap
probabilities, which show the unity of each group, were

Bootstrap probability that shows the unity of each group
Bootstrap probability (%)aSequence

MethodGroup 1

Group 2

Group 3

gyrB

NJ
MLb
MP
NJ
MLb
MP
NJ
MLb
MP
NJ
MLb
MP

100.0
96.1
93.5
99.9
100.0
89.7
98.6
100.0
100.0
99.2
100.0
100.0

100.0
100.0
99.4
98.7
99.4
91.2
98.1
97.0
99.9
99.4
98.5
99.9

75.9
81.0
54.9
99.7
99.4
87.4
100.0
100.0
100.0
100.0
100.0
100.0

NJ
MLb
MP

100.0
100.0
100.0

100.0
98.6
100.0

100.0
100.0
100.0

rpoD

hrpL

hrpS

Tandemly combined
sequence ofall 4 genesa

The bootstrap probability showing the unity of each group, whichappeared in the gene trees (Figs. 1 and 2).
b
For the ML method, the local bootstrap probability (LBP) was estimated by the RELL method (Hasegawa and Kishino 1994; Kishino etal. 1990) with 1000 replications.

all relatively low (87.4–91.2%) for rpoD in the MP
method (Table 3). The gyrB results also showed relatively low unity of Group 3 (54.9–81.0%). However, theother combinations of genes and methods all demonstrated that each group (Groups 1, 2, and 3) is united witha high bootstrap probability (Table 3).

635
Table 4.

Average synonymous distance within each group and between groupsa
Average distance within each group

Average distance between groups

Sequence

Group 1

Group 2

Group 3

Group 1–Group 2Group 1–Group 3Group 2–Group 3

gyrB
rpoD
hrpL
hrpS
Tandemly combined sequenceof all 4 genes

0.123
0.063
0.052
0.125

0.098
0.029
0.048
0.087

0.199
0.033
0.016
0.028

0.575
0.438
0.999
1.179

0.490
0.344
0.856
0.979

0.420
0.219
0.875
0.888

0.086

0.063

0.075

0.690

0.581

0.506

a

Synonymous substitutions were estimated by the method of Nei and Gojobori (1986) using MEGA.

Confirmation by Synonymous Distances. The mean
synonymous distance of gyrB was relatively large(0.199) within Group 3 members, but those of the othercombinations of genes and groups were all small withineach group (Table 4). On the contrary, the mean synonymous distances among groups were all very large. Therefore, the variation within a group was always muchsmaller than those between groups, except for Group 3 in
gyrB.
Monophyly of the Three Groups. As for Group 3 ofthe gyrB gene, members may have started branching earlier than the other groups (Fig. 1), and thus Group 3 may
be less united (Tables 3 and 4). Nevertheless, the OTUs
constituting Group 3 of gyrB are all identical to those ofGroup 3 appeared in the other three gene trees (Figs. 1
and 2).
We therefore concluded that all three groups that had
been confirmed by each gene are monophyletic and thatthese four genes of P. syringae have differentiated into
three groups in the course of evolution. The observationthat the same set of OTUs constitutes each group in all
four genes (Figs. 1 and 2) suggests that these genes had
not experienced any intergroup horizontal gene transfer
within P. syringae but have been stable on and evolved
along with the genome. Therefore, we concluded that
there is no need to separate and compare the pathogenicity-related hrpL and hrpS genes from the housekeeping gyrB and rpoD genes, which had been selected forcomparison with pathogenicity-related genes, but decided to use all four of these genes collectively as anintegrated index for studying the evolutionary course of
the P. syringae genome in the following analyses.
Study of the Branching Order
We could not specify the branching order of the threegroups since the 12 gene trees obtained topologicallyshowed no (3,(1,2)) but both (2,(1,3)) and (1,(2,3)) at aratio of 4:8 (Table 2). The bootstrap probabilities fordetermining the branching orders were all low except for
the NJ analysis for gyrB (94.5%). These results suggestthat such analyses based on each gene are insufficient for

determining the branching order, and we conducted otheranalyses.
Drawing Phylogenetic Trees of the Tandemly Combined Sequence (Concatenated Sequence). As describedin the previous section, the four index genes had notexperienced any intergroup horizontal gene transfer, andthe variation within a group was always smaller than the
difference between groups (Tables 3 and 4). Therefore,
investigation of the group branching order should notcause a serious error even by integrating the sequence
data of the four index genes into one. We drew phylogenetic trees of the tandemly combined sequence using
three methods (NJ, ML, and MP) and compared them.
For each OTU, we integrated its gyrB, rpoD, hrpL,
and hrpS data into one set and conducted an NJ bootstrap
analysis. As in the analyses of each gene, three groups
were shown, all with 100% bootstrap probability (Table3). The OTUs constituting each group were all identicalto those determined by the analyses of each gene. The
branching order of the groups was (1,(2,3)), with a bootstrap probability of 86.9% (Table 2). On the other hand,
the ML tree for the tandemly combined sequence showeda different branching order, (2,(1,3)), with a low local
bootstrap probability of 67.0% (Table 2). The MP tree
also showed a branching order of (2,(1,3)) but, again,
with a low bootstrap probability of 60.9%.
The analyses with tandemly combined sequences did
not give uniform results or high bootstrap probabilities or
could not determine whether the branching order of thethree groups was (2,(1,3)) or (1,(2,3)).
ML Analysis Based on More Realistic Models andParameters. In the second method, we thoroughly investigated the characteristics of the data of the four index
genes, selected models and parameters appropriate foreach datum, analyzed each gene with the ML method,and comprehensively evaluated the four results so obtained. In other words, we comprehensively evaluatedthe four data sets using the TotalML of Molphy version
2.3 (Adachi and Hasegawa 1996) and compared the loglikelihoods among the three topologies concerning thegroup branching order.

636
Table 5. Base composition and ␣/␤ ratio for each codon position of
the four index genes

Sequence
gyrB

rpoD

hrpL

hrpS

Tandemly combined
sequence of all 4 genes

Codon
position

Lengtha

G + Cb

␣/␤c

1st
2nd
3rd
Total
1st
2nd
3rd
Total
1st
2nd
3rd
Total
1st
2nd
3rd
Total

203
203
203
609
170
170
170
510
183
183
183
549
80
80
80
240

0.56
0.36
0.69
0.54
0.73
0.40
0.61
0.58
0.62
0.36
0.66
0.54
0.65
0.42
0.71
0.60

3.7

1st
2nd
3rd
Total

636
636
636
1908

0.63
0.38
0.66
0.56

8.6
2.2
1.7
5.8
2.1
1.6
4.7
1.9
1.9
3.5

2.2
1.8
5.7

a

Insertion/deletion sites were excluded.
Mean values of 31 P. syringae OTUs. The base composition for eachcodon position did not much vary among P. syringae OTUs.
c
The ␣/␤ ratios were optimized for each codon position using NucML(HKY 85 model and rate-heterogeneous model). Because of the small
number of substitutions, ML estimates cannot be obtained for the second codon position of gyrB.
b(a) Selection of Models Based on Characteristics ofSequence Data. We determined the base compositions ofeach gene at three codon positions and found that thecomposition bias varied by codon position (Table 5). The
optimum ␣/␤ ratios also varied by codon position, withthe ␣/␤ ratio of the second codon position being especially low (Table 5). This phenomenon, which had been
known for the mitochondrial genome (Hasegawa andAdachi 1996; Yang 1996), was shown to occur in bacterial genomes in this study. Since any gene is heterogeneous in terms of codon positions, we used the rateheterogeneous model (Hasegawa and Adachi 1996), in
which the ␣/␤ ratio, branch length, and log-likelihood
are separately estimated for each of the three codon positions and the topologies are evaluated by adding thethree log-likelihood values obtained. As for the base substitution model, we adopted the HKY85 model (Hasegawa et al. 1985), since the base composition is biased
to a certain base and the optimum ␣/␤ ratio is not 1 at allcodon positions (Table 5).
(b) ML Analysis Using a Rate-Heterogeneous Model.
The analyses based on bootstrap probability (Table 3)
and synonymous substitution distance (Table 4) showedlarge differences between groups and small variation
within a group, with each group being very homoge-

neous. Thus, we created a user tree for determining thebranching order not of the members constituting eachgroup but of the groups themselves only. Using this user
tree, we compared the log-likelihoods of three topologies, (3,(1,2)), (2,(1,3)), and (1,(2,3)), with TotalML andconducted a comprehensive evaluation of the groupbranching order.
For gyrB, a comprehensive evaluation of the analysesof each codon position resulted in an ML tree of (2,(1,3))(Table 6). A rpoD analysis also showed (2,(1,3)) to be
the most likely. A comprehensive evaluation of these twodata sets also supported (2,(1,3)) most. The ML trees for
hrpL and hrpS were also (2,(1,3)) (Table 6). A comprehensive evaluation of hrpL and hrpS data also supported(2,(1,3)).
A comprehensive evaluation of these four genes
strongly supported (2,(1,3)) (Table 6). On the other hand,
the topology of (3,(1,2)) was rejected at the 5% level ofsignificance, with a log-likelihood lower by 12.6 ± 6.2(0.87% BP). The (1,(2,3)) topology was not rejected,
although its bootstrap probability was very low, 9.9%. A
rate-heterogeneous model analysis of the tandemly combined sequence, in which the sequence data of the fourindex genes were concatenated, also strongly supported(2,(1,3)) and rejected (3,(1,2)), with a log-likelihood
lower by 14.5 ± 6.8 (0.29% BP). (Table 6).
An amino acid sequence analysis with the JTT-F
model using the ProtML and TotalML programs (Adachi
and Hasegawa 1996) also showed (3,(1,2)) to have theworst likelihood and the lowest bootstrap probability(Table 7). (2,(1,3)) was comprehensively best in likelihood, but the likelihood difference with (1,(2,3)) was notas large as that shown by the DNA sequence analyses(Table 6).
Branching Order of the Three Groups. Among thethree topologies for the group branching order, (3,(1,2))
was statistically rejected by the TotalML analysis of
DNA sequences (Table 6). None of the analyses conducted for each gene supported this topology either(Table 2). Therefore, (3,(1,2)) should be excluded fromthe branching order candidates. On the other hand,(2,(1,3)) was most supported by both the DNA sequenceand the amino acid sequence analyses (Tables 6 and 7).
The order (1,(2,3)) showed a worse log-likelihood than(2,(1,3)) but could not be totally excluded (Tables 6 and
7). We concluded that the group branching order of P.
syringae should not be determined as (2,(1,3)) or(1,(2,3)) with only the data used in this study. The difficulty may be attributable to these three groups having
continuously branched within a very short period in theevolutionary time scale and/or to the inadequacy of theselection of the outgroups. More P. syringae relatives
and new indices should be analyzed to accumulate data,
and analytical methods should be further investigated todetermine the branching order.

637
Table 6.

Comprehensive evaluation of tree topologies (group branching order) based on NucML and TotalML analyses of the four index genes(3,(1,2))Codon
position(2,(1,3))(1,(2,3))

Length

⌬la

BPb

⌬la

BPb

⌬la

BPb

1st
3rd
Total
1st
2nd
3rd
Total

207
207
414
175
175
175
525
939

−1.0 ± 1.7〈−2488.5〉−0.3 ± 1.7〈−596.7〉−2.7 ± 2.6−4.0 ± 3.2−5.0 ± 4.1−4.6 ± 4.4

0.01210.47420.31840.82740.05680.02730.07780.1282〈−825.7〉−0.7 ± 1.4〈−3314.8〉−1.7 ± 1.7〈−546.0〉〈−1324.0〉〈−2468.4〉〈−5783.5〉

0.65670.12100.43130.08400.86660.65470.77110.7289−1.0 ± 1.7−0.2 ± 1.8−0.5 ± 2.5−1.7 ± 1.7−2.7 ± 2.6−1.8 ± 4.2−4.5 ± 5.2−4.7 ± 5.8

0.33120.40480.25030.0886
0.0766
0.31800.15110.1429

1st
2nd
3rd
Total
1st
2nd
3rd
Total

Total for hrpL and hrpS

194
194
194
582
80
80
80
240
822

−3.0 ± 1.8−0.2 ± 0.6−0.3 ± 1.1−3.1 ± 2.2−1.9 ± 2.1−2.9 ± 2.4−1.2 ± 2.1−4.7 ± 3.8−7.8 ± 4.4

0.01760.27570.33930.02780.06820.03140.19240.01440.0034〈−897.0〉〈−709.3〉−0.4 ± 0.9〈−3084.8〉〈−512.2〉〈−376.3〉−1.3 ± 2.0〈−1760.6〉〈−4845.4〉0.8014
0.59850.1036
0.72180.79840.74170.13070.72900.8169−1.8 ± 2.1−0.2 ± 0.6〈−1478.1〉−1.6 ± 2.2−1.9 ± 2.1−2.1 ± 2.9〈−870.7〉−2.6 ± 3.6−4.2 ± 4.2

0.18100.12580.55710.25040.13340.22690.67690.25660.1797

Total for all 4 genes

1761−12.6 ± 6.2d

0.0087〈−10628.9〉

0.8926−8.9 ± 7.2

0.0987

656
656
656
1968

−8.1 ± 4.8−5.1 ± 3.7−1.4 ± 2.9−14.5 ± 6.8d

0.01120.02030.17430.0029〈−3032.5〉〈−2230.9〉〈−6548.3〉〈−11811.7〉

0.83550.86770.52030.9261−5.5 ± 5.5−4.4 ± 4.0−0.8 ± 3.1−10.7 ± 7.5

0.15330.11200.30540.0710

Sequence
gyrBc

rpoD

Total for gyrB and rpoD
hrpL

hrpS

Tandemly combined sequenceof all 4 genes

1st
2nd
3rd
Total

a

The log-likelihood of the ML tree is given in angle braces, and thedifferences in log-likelihood of alternative topologies from that of the
ML tree (⌬l) are shown with their SEs (following ±), which were estimated by Kishino and Hasegawa’s formula (Adachi and Hasegawa 1996).
b
The bootstrap probabilities (BP) were estimated by the RELL method(Hasegawa and Kishino 1994; Kishino et al. 1990) with 10,000 replications.

cBecause of the small number of substitutions, ML estimates cannot be
obtained for the second codon position of gyrB, and this position was
not used in the analysis.
d
The relationship of (3,(1,2)) was rejected at the 5% level of significance.

Table 7. Comprehensive evaluation of tree topologies (group branching order) based on ProtML (JTT-F model) and TotalML analyses of the fourindex genes(3,(1,2))(2,(1,3))(1,(2,3))Sequence

Length

⌬la

BPb

⌬la

BPb

⌬la

BPb

gyrB
rpoD
Total for gyrB and rpoD

207
175
382

−2.6 ± 2.6−3.4 ± 3.1−4.8 ± 4.0

0.04780.05050.0110〈−1182.9〉−1.9 ± 3.8−0.7 ± 3.8

0.59980.30500.4325−1.2 ± 3.0〈−1207.3〉〈−2391.4〉

0.35240.64450.5565hrpL
hrpS
Total for hrpL and hrpS

193
80
273

−3.0 ± 1.6−2.2 ± 2.4−4.4 ± 2.9

0.00400.03980.0021〈−1610.5〉−0.8 ± 3.6〈−2510.2〉

0.97240.38740.7137−2.9 ± 1.7〈−898.9〉−2.2 ± 1.7

0.0236
0.57280.2842655
655−8.4 ± 5.0−8.7 ± 5.0

0.00010.0003〈−4902.3〉〈−5183.9〉

0.58620.7695−1.4 ± 3.5−4.9 ± 6.6

0.41370.2302

Total for all 4 genes
Combined for all 4 genesaThe log-likelihood of the ML tree is given in angle braces, and the differences in log-likelihood of alternative topologies from that of the ML tree(⌬l) are shown with their SEs (following ±), which were estimated by Kishino and Hasegawa’s formula (Adachi and Hasegawa 1996).
b
The bootstrap probabilities (BP) were estimated by the RELL method (Hasegawa and Kishino 1994; Kishino et al. 1990) with 10,000 replications.

Construction of a Phylogenetic Tree Expressing the
Genome Evolution of P. syringae
We attempted to construct a phylogenetic tree (genometree) that expresses the genome evolution of P. syringae

by collectively using the data on all four index genes. We
used the tandemly combined sequence of the four genes
as indices based on the reasons described in the previous
section. Since we could not determine the group branching order with the analyses described in the previous

638

Fig. 3. ML tree expressing the evolution of the P. syringae genome,
based on the tandemly combined sequence (concatenated sequence) of
the four index genes (gyrB, rpoD, hrpL, and hrpS) of 31 P. syringae
OTUs excluding outgroups (PAE and ECO), constructed using NucML[HKY85 model (Hasegawa et al. 1985); ␣/␤ ⳱ 4.5, InL ⳱ −6694.01](see text for details). The group branching order of this tree is either(2,(1,3)) or (1,(2,3)), with (2,(1,3)) being much more likely (see Tables
6 and 7 and the text). An identical topology was found by MP analysis.
Horizontal branch lengths are proportional to the estimated number ofnucleotide substitutions, and local bootstrap probabilities (as percentages), as determined for 1000 resamplings, are given above the internalbranches. Abbreviations for strains (OTU names) are as listed in Table
1. Stars indicate the strains which are capable of producing phaseolotoxin and have the identical argK gene on their genomes. Each strainis labeled with the pathovar name to which it belongs and the familyname of the host plant from which it was isolated. The number inparentheses after each pathovar name indicates the number of strainsused which belong to that pathovar and are included in that clade.

sections [although (2,(1,3)) is most likely], we excluded
the data on outgroups, E. coli (ECO) and P. aeruginosa(PAE), and drew an unrooted tree with only P. syringae
data.
The resulting ML tree clearly showed distinction
among Groups 1, 2, and 3, all with 100% bootstrap probability (Fig. 3). The topologies within each group were
all identical to those of the ML tree determined from thetandemly combined sequences of 33 OTUs, including theoutgroups (ECO and PAE), which is described in theprevious section. The MP tree also showed clear separation of Groups 1, 2, and 3, all with 100% bootstrap
probability (data not shown). The topologies within eachgroup were all identical to those of the ML tree (Fig. 3).
The NJ tree also showed Groups 1, 2, and 3, all with
100% bootstrap probability (Fig. 4). The topologies
within each group were all identical to those of the NJ
tree determined from 33 OTUs, including the outgroups(see the previous section). This correspondence is likely
attributable to each group being constituted by a suffi-

639

structed an unrooted tree with P. syringae data (31OTUs) only and assumed a tentative root for the unrooted tree based on the likeliest (2,(1,3)) topology. Information concerning various phenotypes and pathogenicity-related genes was added to the genome tree
obtained (Fig. 3), whose evolutionary mechanisms in P.
syringae are discussed below.
Relationship with the Pathovar to Which a
Strain Belongs

Fig. 4. NJ tree expressing the evolution of the P. syringae genome,
based on the tandemly combined sequence of the four index genes,
constructed using the distance matrix derived from the two-parameter
method (Kimura 1980) (see the text and the legend to Fig. 3 for details).
The group branching order of this tree is either (2,(1,3)) or (1,(2,3)),
with (2,(1,3)) being much more likely (see Tables 6 and 7 and text).
The branching order of the OTUs shallower than AR1 in Group 2 isslightly different from those of the MP and ML trees (Fig. 3). Horizontal branch lengths and bootstrap probabilities are as described in thelegend to Fig. 1. Abbreviations for strains (OTU names) are as listed inTable 1.

cient number of OTUs (around 10), thus stabilizing the
topological estimation. A comparison of newly determined NJ, ML, and MP trees (Figs. 3 and 4) showed aslight difference of OTU branching order between NJ
tree and the others in Group 2 but totally identical topologies concerning Groups 1 and 3. This topologicaldifference in Group 2 was insignificant for the major
conclusions of this study.

Discussion
With consideration of the results obtained, we collectively used the data on all four index genes (gyrB, rpoD,
hrpL, and hrpS) and created a genome tree that expresses
the evolutionary course of the genome in the final section
under Results (Figs. 3 and 4). Since we could not determine the group branching order (see Results), we con-

We located the pathovar name to which each strain belongs in the genome tree (Fig. 3). Pathovar tomato (OTU
name of the corresponding strains: TO1), pv. maculicola(MA1, MA2), pv. lachrymans (LA1), pv. morsprunorum(MP1), pv. syringae (SY7), pv. actinidiae (AC30), andpv. theae (TH2, TH3) were located in Group 1; pv. aceris (AR1), pv. aptata (AP1), pv. japonica (JA1), pv.
syringae (SY1, SY2, SY5), pv. pisi (PI1, PI2) were inGroup 2; and pv. myricae (MY1), pv. eriobotryae (ER1),
pv. morsprunorum (MP2, MP3), pv. tabaci (TB1), pv.
lachrymans (LA2), pv. castaneae (CA1), pv. phaseolicola (PA1, PU4), pv. glycinea (GL1), pv. mori (MR1,
MR2, MR6), and pv. broussonetiae (BR1) were in Group
3. A phylogenetic analysis based on the hrpJKL-region
sequence (Cournoyer et al. 1996) had confirmed that pv.
pisi and pv. syringae are close relatives and agreed withthe results of this study (Figs. 3 and 4). PCR-RFLP
analysis of the rrn operon (Manceau and Horvais 1997)
and AFLP and RAPD analyses (Clerc et al. 1998) had
confirmed the close relationship between pv. tomato andpv. maculicola, also agreeing with the results of thisstudy (Figs. 3 and 4).
Three pathovars, pv. lachrymans, pv. morsprunorum,and pv. syringae, are distributed over two groups (Figs.
3 and 4). The pathotype strains of pv. lachrymans (LA1)and pv. morsprunorum (MP1) are included in Group 1,
but all the other OTUs (LA2 for pv. lachrymans, MP2
and MP3 for pv. morsprunorum) representing Japanesestrains used (Table 1) belong to Group 3. For pv. syringae, only a Japanese citrus strain (SY7) (Shigeta and
Nakata 1995) belongs to Group 1, while all the other
OTUs, representing the pathotype strain (SY1) and another three Japanese strains (SY2 and SY5), are includedin Group 2.
The cause for these three pathovars being distributed
over two groups and being genetically nonuniform may
be associated with incorrect or incomplete pathovar definition or strain identification (Bradbury 1986; Rudolph
1995; Young et al. 1992). Various indices suggest that
pv. syringae is especially heterogeneous and is a collection of multiple kinds of strains (Cameron 1962; Dennyet al. 1988; Legard et al. 1993; Young 1991; Young et al.
1992), which may represent different pathovars or evendifferent species (Young 1991). On the other hand, it isalso suggested that nine pathovars (pv. aceris, pv. aptata,
pv. atrofaciens, pv. dysoxyli, pv. japonica, pv. lapsa, pv.
panici, pv. papulans, and pv. pisi) may be synonyms of

640

pv. syringae (Gardan et al. 1991, 1994; Young 1992;
Young et al. 1992). Of these nine pathovars, this study
tested four [pv. aceris (AR1), pv. aptata (AP1), pv. japonica (JA1), and pv. pisi (PI1, PI2)] and revealed that
all of them and pv. syringae (SY1, SY2, SY5) formed
one tight cluster, Group 2 (Figs. 3 and 4). Our results
agree with studies based on the DNA–DNA hybridization technique (Gardan et al. 1994) and may provide
gene-level support for the synonymy of these pathovars.
As shown above, the pathovar system of P. syringae iscomplicated and confused in terms of classification(Bradbury 1986; Rudolph 1995; Young et al. 1992).
Thus, the classification of P. syringae should be carefully investigated by testing many strains of diverse histories and by conducting and cross-checking gene-level
analyses and phenotype studies.
Of the 16 pathovars other than pv. lachrymans, pv.
morsprunorum, and pv. syringae, 8 were tested for two
or more strains (Table 1); pv. maculicola (number ofstrains tested, five; OTU names, MA1 and MA2), pv.
actinidiae (five; AC30), pv. theae (six; TH2 and TH3),
pv. pisi (two; PI1 and PI2), pv. phaseolicola (eight; PA1
and PU4), pv. mori (six; MR1, MR2, and MR6), pv.
tabaci (three; TB1), and pv. glycinea (two; GL1). As far
as these eight pathovars were concerned, all strains thatbelong to the same pathovar were always closely locatedin the genome tree (Figs. 3 and 4), suggesting their homogeneity. Pathovar actinidiae is especially homogeneous as we reported previously (Sawada et al. 1995a,
1996, 1997a), since the sequences of the four index
genes investigated in this study were identical in all fivestrains tested.

Relationship Between Strains and Plant Sources(Host Plants)
We then located the family names of host plants, fromwhich the strains were isolated, in the genome tree (Fig.
3). Group 1 includes many diverse host plant families,
such as Solanaceae (OTU name of the correspondingstrains: TO1), Brassicaceae (MA1 and MA2), Cucurbitaceae (LA1), Rosaceae (MP1), Rutaceae (SY7), Actinidiaceae (AC30), and Theaceae (TH2 and TH3), andshowed no special characteristics or correlation between
the phylogenetic relationship of OTUs and that of their
host plants.
The host range of pv. syringae included in Group 2 isconsidered to be very wide, including Pisum sativum,
Triticum aestivum, and Beta vulgaris (Bradbury 1986;
Young 1991), which are also hosts for pv. pisi, pv. japonica, and pv. aptata of the same group, respectively(Bradbury 1986). Thus, Group 2 members appear to havecommon host plants with pv. syringae, but this interpretation must be carefully examined by further studying thedefinition of pv. syringae as discussed in the previous
section.

In Group 3, pv. mori (MR1, MR2, MR6) and pv.
broussonetiae (BR1), which infect Moraceae plants, areclosely located, as are pv. phaseolicola (PA1, PU4) andpv. glycinea (GL1) from Fabaceae and pv. morsprunorum (MP2, MP3) and pv. eryobotryae (ER1) from Rosaceae, respectively (Figs. 3 and 4). The three strains of
pv. tabaci (TB1) tested were from Nicotiana tabacum,
whose host range also includes Cucumis sativus (Bradbury 1986), and are located in a tree near pv. lachrymans(LA2) isolated from Cucumis sativus. Therefore, we may
conclude that in Group 3 there are four subgroups, whose
members were isolated from host plants that are closely
related to each other.
Groups 2 and 3 showed a correlation between thephylogenetic relationships of OTUs and those of their
host plants. To study these relationships in terms of coevolution, we should further elucidate the phylogenetic
relationships of host plants and the molecular mechanisms of pathogenicity.
Evolutionary Stability of the hrp Gene Cluster Within
P. syringae
The hrp/avr genes are considered to exist in all P. syringae pathovars and to be involved in the pathogenicity
and the determination of host range (reviewed by Baker
et al. 1997; Gopalan and He 1996). Among these genes,
the ones that are involved in the fundamental processes
such as gene expression control and secretion of geneproducts form a supraoperon clustering structure (the socalled “hrp gene cluster”) on the genome. The cluster isbelieved to contain at least 25 genes that are organized
into seven separate transcription units. The hrpL and
hrpS genes that were selected to represent pathogenicityrelated genes in this study are located near the left andright ends of the hrp gene cluster, respectively, and are
involved in the expression control of the hrp/avr genes.
To investigate the evolutionary mechanism of the hrp
gene cluster, we compared the phylogenetic analyses forthe inside of the hrp gene cluster (hrpL and hrpS; representing the cluster) and those for the outside section(gyrB and rpoD; representing the genome).
NJ, ML, and MP analyses showed that the gyrB and
rpoD genes located outside of the cluster had both differentiated into three independent groups, as had thehrpL and hrpS genes located in the cluster (Tables 2, 3,
and 4). The OTUs constituting each group were identicalfor genes in and out of the cluster. Total ML analysis
with a rate-heterogeneous model showed the highest loglikelihood when the branching order of the three groups
was (2,(1,3)) for both gyrB and rpoD (Table 6). Similarly, (2,(1,3)) was the ML tree for both hrpL and hrpS.
Therefore, the data for both the outside and the inside of
the cluster showed the same tendency. We, thus, concluded that the hrp gene cluster of the strains tested inthis study had not experienced any intergroup horizontal
gene transfer within P. syringae, and the hrp gene cluster
and the P. syringae genome, on which the cluster is641

located, likely followed the same course of evolution. It
was recently postulated that hrp genes may have been
introduced into the ancestor of P. syringae from an unknown enteric animal pathogen by horizontal gene transfer (Brown et al. 1998). At least after that event, theyshould have been stable on the P. syringae genome.
Pathogenicity-related genes, whose expression is controlled and/or whose products are secreted outside of thebacteria by the function of hrp genes, were found also toexist on non-hrp gene cluster sections of the genome andplasmids (Baker et al. 1997; Gopalan and He 1996; Yuan
and He 1996). Thus, to understand fully the evolution ofpathogenicity associated with the hrp gene cluster, weshould systematically analyze not only the so-called corehrp gene cluster itself, which is the regulation and secretion system, but also the data for various pathogenicity-related genes under its control.

Expansion of the argK (argK–tox Gene Cluster)Distribution in P. syringae
Phaseolotoxin is a non-host-specific toxin that is active
not only in plants but in microorganisms including E.
coli (reviewed by Rudolph 1990). The enzyme that is thetarget of the toxin is anabolic ornithine carbamoyltransferase (OCTase; EC 2.1.3.3) in the biosynthetic pathway
of arginine. This means, in turn, that the biosynthesis of
L-citrulline from L-ornithine and carbamoylphosphate isinhibited by the toxin. This is thought to be the cause ofboth the accumulation of L-ornithine and the deficiencyof L-citrulline and L-arginine (Mitchell and Bieleski
1977; Mitchell 1984; Patil et al. 1970; Rudolph and Stahmann 1966).
Pathovar phaseolicola, which produces phaseolotoxin, has both phaseolotoxin-sensitive OCTase (SOCT)
and phaseolotoxin-resistant OCTase (ROCT). It is
known that pv. phaseolicola is resistant to phaseolotoxin
produced by itself since ROCT is not inhibited byphaseolotoxin but remains active (Ferguson et al. 1980;
Jahn et al. 1985; Staskawicz et al. 1980; Templeton et al.
1986). The gene that codes for ROCT, argK (Hatziloukas and Panopoulos 1992; Mosqueda et al. 1990), is
contained in the tox cluster (Hatziloukas and Panopoulos
1992; Peet, and Panopoulos 1987), which is involved in
the biosynthesis of phaseolotoxin (Peet et al. 1986;
Zhang et al. 1993).
Phaseolotoxin, which was first discovered as a toxinof pv. phaseolicola (Rudolph 1990), is also produced by
pv. actinidiae and has been proved actually to cause
chlorosis in pv. actinidiae-infected kiwifruit leaves(Tamura et al. 1989, 1997). We also confirmed that all
strains of these two pathovars tested (13 strains that are
expressed with three OTU names: AC30, PA1, and PU4)
(Table 1) produced phaseolotoxin and had an argK geneon their genome, using Southern blot analysis (Sawada etal. 1995a, 1997a; Sawada, manuscript in preparation).

On the contrary, we found that the other 43 strains usedin this study (Table 1), which do not belong to these twopathovars, were not capable of producing phaseolotoxin
and did not have the argK gene (Sawada et al. 1995a,
1997a; Sawada, manuscript in preparation). Thus, theproductivity of phaseolotoxin and the possession of theargK gene are shown at only two separate points in the
genome tree (Fig. 3): Group 1 (AC30; pv. actinidiae) andGroup 3 (PA1 and PU4; pv. phaseolicola). It is known
that the argK–tox gene cluster is located on the genome(Peet et al. 1986; Sawada, manuscript in preparation;
Zhang et al. 1993). The following paragraphs discuss thereason for the argK–tox gene cluster on the genome, noton a conjugative plasmid, being distributed over two
phylogenetic groups (Groups 1 and 3) and the expansion
mechanisms of its distribution.
Hatziloukas and Panopoulos (1992) analyzed argK of
pv. phaseolicola isolated from Phaseolus vulgaris in the
United States and found that the GC content of the argK
gene and that of the whole genome of pv. phaseolicola
were very different. Thus, they hypothesized that theargK gene originated from another prokaryote. Narumi
and Takikawa (1996) compared the argK–tox gene cluster structures of pv. phaseolicola and pv. actinidiae using
restriction enzyme maps and found that the structures
were similar for the inside section of the cluster but weredifferent for the outside between the two pathovars. We
revealed that the 13 strains of pv. phaseolicola and pv.
actinidiae used (Table 1) have argK, whose sequences(D86356) are completely identical to each other, without
a single synonymous substitution (Sawada et al. 1995a,
1997a; Sawada, manuscript in preparation), also coincidewith that (M94049) of pv. phaseolicola isolated in the
United States (Hatziloukas and Panopoulos 1992) andare quite similar (only three substitutions exist) to that
(X55520) of pv. phaseolicola isolated in Mexico(Mosqueda et al. 1990). On the other hand, we found thatthe genomes of pv. phaseolicola and pv. actinidiae belong to different and separate phylogenetic groups(Groups 3 and 1, respectively) (Tables 3 and 4, Figs. 3
and 4).
Therefore, the following three hypotheses may be derived for the origin and expanding distribution of argK or
the argK–tox gene cluster. (1) argK (argK–tox gene cluster) was introduced by horizontal gene transfer from theoriginal organism into the genome of a common ancestorof pv. actinidiae (Group 1) and pv. phaseolicola (Group3). Afterward, as the ancestor evolved into pv. actinidiae
and pv. phaseolicola, the argK gene took the corresponding evolutionary course along with and stably on the
genome (no horizontal gene transfer of argK occurred
after the pathovars separated). (2) After pv. actinidiae
and pv. phaseolicola separated from the common ancestor, argK was introduced by horizontal gene transferfrom the original organism into either pv. actinidiae or
pv. phaseolicola, from which the gene was introduced by

642

the second horizontal gene transfer into the other. (3)After pv. actinidiae and pv. phaseolicola separated, argK
was introduced from the original organism into pv. actinidiae and pv. phaseolicola by two independent horizontal gene transfers. We examined these three hypotheses based on the results of this study.
The tandemly combined sequence (index for genome
evolution) of gyrB, rpoD, hrpL, and hrpS was used toderive the synonymous distance and the standard error
between AC30 (pv. actinidiae) and PA1 (pv. phaseolicola isolated from Phaseolus vulgaris or Pueraria lobata) to be 0.5316 ± 0.0463. The distance between AC30
and PU4 (pv. phaseolicola isolated from Pueraria lobata) was 0.5308 ± 0.0462, a similar value. On the contrary, there is no synonymous substitution in the argK
gene between pv. actinidiae (AC30) and pv. phaseolicola (PA1 and PU4). Thus, a t test between pv. actinidiae
and pv. phaseolicola for the synonymous distances of thetandemly combined sequence and argK showed t ⳱
0.53/0.046 ⳱ 11.5, which is highly significant. This result suggests that argK of these two pathovars had taken
evolutionary courses different from those of the four index genes selected to represent their genomes. This andother analyses of ours (Sawada et al. 1995a, 1997a;
Sawada, unpublished data) revealing the lack of argK on
the genomes of the 43 strains used other than pv. actinidiae or pv. phaseolicola strongly suggest that the argK
gene (argK–tox gene cluster) experienced horizontal
gene transfer after the pathovars had separated, thus
showing a base substitution pattern extremely different
from that of the noncluster region of the genome. Hence,
the first of our three hypotheses mentioned above is rejected. The argK gene was likely introduced very recently in terms of evolutionary time scale, either (1) byhorizontal gene transfer from the original organism to pv.
actinidiae or pv. phaseolicola, and from each to the otherby the second transfer, or (2) by two independent horizontal gene transfers from the original organism to pv.
actinidiae and pv. phaseolicola.
To verify these two hypotheses, we should (1) test
more strains of pv. actinidiae and pv. phaseolicola withdiverse histories, (2) compare the DNA sequences of the
tox cluster other than the argK region and the structuresof the genome near the cluster boundary, (3) identify theoriginal organisms from which the argK–tox gene cluster
was derived, and so on. When and how the gigantic“pathogenicity island”-like structure of the argK–tox
gene cluster was formed is another important topic.
This study revealed that the P. syringae strains useddifferentiated into three different monophyletic groups.
Our phylogenetic analysis of the evolutionary mechanisms of argK (argK–tox gene cluster) showed that it had
expanded its distribution over two pathovars by horizontal gene transfer. On the other hand, the hrp gene cluster
had not experienced horizontal gene transfer between
groups of P. syringae but had been stable on the genome.

We are now conducting studies from other viewpoints
based on the results of this study: we will further investigate the origins and evolutionary mechanisms of theargK–tox gene cluster and other pathogenicity-related
genes by analyzing the frequency of codon usage, comparing the genome structures, and conducting verification tests concerning the evolutionary mechanisms, suchas horizontal gene transfer and genome rearrangement.
Acknowledgments. We thank Masami Hasegawa, Satoshi Oota,
Naoko Takezaki, Satoshi Yamamoto, and Yuichi Takikawa for theirhelpful suggestions and Koushi Nishiyama, Mamoru Sato, Yuichi
Takikawa, Takanori Miyoshi, Yatsuka Nishi, Susumu Shigeta, and Kazuo Takanashi for providing the bacterial strains used in this study.

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