J Mol Evol (2002) 54:437–457
DOI: 10.1007/s00239-001-0032-y© Springer-Verlag New York Inc. 2002

A Phylogenomic Study of the OCTase Genes in Pseudomonas syringae
Pathovars: The Horizontal Transfer of the argK–tox Cluster and the
Evolutionary History of OCTase Genes on Their Genomes
Hiroyuki Sawada,1 Shigehiko Kanaya,2 Masataka Tsuda,3 Fumihiko Suzuki,1 Kozi Azegami,1 Naruya Saitou4
1

National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan
Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
3
Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan
4
National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
2

Received: 22 May 2001 / Accepted: 26 September 2001

Abstract. Phytopathogenic Pseudomonas syringae issubdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Basedon the results of a phylogenetic analysis of four genes(gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999)
showed that the ancestor of P. syringae had diverged into
at least three monophyletic groups during its evolution.
Physical maps of the genomes of representative strains ofthese three groups were constructed, which revealed thateach strain had five rrn operons which existed on one
circular genome. The fact that the structure and size ofgenomes vary greatly depending on the pathovar showsthat P. syringae genomes are quite rich in plasticity andthat they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content
at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involvedin phaseolotoxin synthesis (argK–tox cluster) expanded
its distribution by conducting horizontal transfer onto the
genomes of two P. syringae pathovars (pv. actinidiae
and pv. phaseolicola) from bacterial species distantly
related to P. syringae and that its acquisition was quite
recent (i.e., after the ancestor of P. syringae diverged
into the respective pathovars). Furthermore, the results ofa detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which ispresent within the argK–tox cluster, revealed the plausible process of generation of an unusual composition of
the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene(equivalent to the orthologue of arcB of P. aeruginosa)
and an anabolic OCTase gene (argF), which must havebeen formed by gene duplication, have first been present
on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has
diverged into the respective pathovars; the foreignoriginated argK–tox cluster has horizontally transferred
onto the genomes of pv. actinidiae and pv. phaseolicola;and hence two copies of only the anabolic OCTase genes(argK and argF) came to exist on the genomes of thesetwo pathovars. Thus, the horizontal gene transfer and the
genomic rearrangement were proven to have played an
important role in the pathogenic differentiation and diversification of P. syringae.
Key words: Pseudomonas syringae — argK–tox
cluster — Ornithine carbamoyltransferase — Genome
plasticity — Physical map — Codon usage — Molecularphylogeny — Gene duplication — Paralogue — Horizontal gene transfer

Introduction

Correspondence to: H. Sawada; email: sawada@affrc.go.jp

Pseudomonas syringae, which is a phytopathogenic bacterium, is known to be rich in diversity and has hitherto

438

been subdivided into about 50 pathovars, mainly withregard to phytopathogenicity and host ranges (Bradbury
1986; Dye et al. 1980; Rudolph 1995; Young et al.
1992). It is essential to identify the composition of pathogenicity-related genes of P. syringae, as well as the process in which they have undergone variations and functional differentiation, to understand the nature of thisconspicuous pathogenic differentiation. In addition, there
are cases in which different types of pathovars (pv.) affect the same plant species and/or cause infected plants
to exhibit similar disease symptoms, so it is suspectedthat the same pathogenicity-related genes exist in various
pathovars as a result of interpathovar horizontal transfer(Hatziloukas and Panopoulos 1992; Sawada et al. 1997,1999). Genomes of phytopathogenic bacteria can be considered “vehicles of genes” for the passage of pathogenicity-related genes by horizontal transfer. Accordingly,
if the course of genome evolution is identified, the courseof the divergence and horizontal transfer of pathogenicity-related genes may be clarified by comparing genomes
and pathogenicity-related genes in view of the process ofevolution.
Thus, we tried to clarify the nature of the pathogenic
differentiation of P. syringae, mainly through phylogenetic analysis. A comprehensive phylogenetic analysis offour genes (gyrB, rpoD, hrpL, and hrpS) of 56 strains
belonging to 19 pathovars showed that the ancestor of P.
syringae (the common ancestor prior to the divergence
into pathovars) had diverged into at least three monophyletic groups (Groups 1 to 3) (Sawada et al. 1999). In
addition, the results suggested that the gene cluster involved in phaseolotoxin synthesis (argK–tox cluster)
may have expanded its distribution onto the genomes of
two pathovars (P. syringae pv. actinidiae and P. syringae
pv. phaseolicola) by conducting horizontal transfer(Sawada et al. 1997, 1999). Pathovar actinidiae is known
as a causative agent of bacterial canker of kiwifruits, andpv. phaseolicola as a causative agent of halo blight disease of beans, both of which cause a disease symptom(chlorosis) on the leaves of the host plants. Because
phaseolotoxin is a virulence factor that causes chlorosis
by inhibiting the enzymatic activity in host plants(Bender et al. 1999), we suspected that the argK–tox
cluster may have been directly involved in the pathogenic differentiation by conducting horizontal transfer
among pathovars.
After the whole-genome sequence of Haemophilus
influenzae was determined in 1995, numerous genome
sequencing projects were implemented for prokaryotes,
and genome sequences of more than 50 prokaryotes havebeen reported so far (e.g., see http://www.tigr.org/tdb/).
The progress of such genome sequencing projects gave
rise to genomics, study to elucidate the general outlookof life by thoroughly clarifying the whole (genomes) inaddition to the pieces (genes) (Galperin et al. 1999; Watanabe et al. 1997).

Has the argK–tox cluster existing on genomes actually experienced horizontal transfer? How has this affected the evolution and diversification of P. syringae?
To answer these questions, it is essential to conduct theabove-mentioned genomic analysis to supplement phylogenetic analysis. Combination of these two approaches
can be called “phylogenomic” analysis (proposed by Eisen 1998). In this study, we analyzed the argK–tox cluster from a phylogenomic approach, which was analyzed
from a phylogenetic approach in our previous study(Sawada et al. 1999). The procedure of the genomic
analysis conducted in this study was as follows: the genomic structure of representative strains of the respective
monophyletic groups (Groups 1 to 3) of P. syringae was
identified by I-CeuI fragment analysis; physical maps ofthese strains were constructed, onto which the argK–tox
cluster was mapped; and then an interpathovar comparison was made.
If the argK–tox cluster has in fact experienced horizontal transfer, what is its initial origin, pv. actinidiae,
pv. phaseolicola, or a bacterial species other than P.
syringae (i.e., of foreign origin)? Rigorous analyses were
thereby conducted using argK [a gene that encodes anabolic ornithine carbamoyltransferase (anabolic OCTase;
EC 2.1.3.3)], which is present within the argK–tox cluster, as a clue: (1) a phylogenetic tree was constructedusing the information on all the already-reported OCTase
genes to clarify the evolutionary history of OCTase
genes, the “course of argK evolution” within this evolutionary history was identified, and then whether or not
this course agrees with the “course of genomic evolutionof P. syringae” was examined; and (2) as the codon
usage pattern and GC content at the codon third position
are known as highly effective criteria for determining
whether or not genes are of foreign origin (Kanaya et al.
1996a, b, 1997), they were used as indices to compare
argK with other genes on the P. syringae genomes.
This particular anabolic OCTase, called “ArgK,” isknown to play an extremely important role in the survival and pathogenicity of P. syringae (Jahn et al. 1985;
Rudolph 1990; Staskawicz et al. 1980; Templeton et al.
1986). The final analysis conducted in this study focusedon the clarification of the relationship between the courseof OCTase evolution and the evolution and diversification of P. syringae by reproducing the evolutionary history of OCTase genes unfolded on the P. syringae genomes.
The results of these analyses showed that genomic
rearrangements occurred on a large scale in the processof the divergence of the ancestor of P. syringae into therespective pathovars. In addition, our hypothesis that the“argK–tox cluster is of foreign origin and has expanded
its distribution by conducting horizontal transfer” wasconfirmed to be consistent and unequivocal throughout
the phylogenomic analyses. Furthermore, the plausible
process of generating an unusual OCTase gene compo-

439
Table 1.

Strains of Pseudomonas species used in this study

SpeciesP. aeruginosa
P. fluorescens
P. putida
P. syringae

Groupa1

2

3

Pathovar

Strain designationb

Geographic originc

Sourcebactinidiae
maculicolatheaemorsprunorum
lachrymans
syringae
pisi
aptatajaponica
acerisphaseolicola
phaseolicola
glycineatabacimori

PAO 1
MAFF 302208T
IAM 1236
KW-11PT
MAFF 302264PT
PT1PT
MAFF 302280PT
MAFF 302278PT
MAFF 302155T
MAFF 302269PT
MAFF 302253PT
MAFF 301072PT
MAFF 302273PT
MAFF 302282PT
KUZ 1
MAFF 302260PT
MAFF 302270PT
MAFF 302279PT

?
UK
USA
Shizuoka, JapanNew Zealand
Shizuoka, Japan?
USAUK
New ZealandUSA
Tochigi, Japan
USA
CanadaIbaraki, JapanNew Zealand
Hungary
Hungary

J. KatoMAFF
IAM
Y. Takikawa
MAFF
Y. Takikawa
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
MAFF
M. Sato
MAFF
MAFF
MAFF

a

The ancestor of P. syringae (the common ancestor prior to the divergence into pathovars) had so far diverged into at least three monophyletic
groups (Groups 1 to 3) in the process of evolution (Sawada et al. 1999).
b
IAM—IAM Culture Collection, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan; MAFF—
Microorganisms Section of the MAFF Gene Bank, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan; J. Kato—
Junichi Kato, Faculty of Engineering, Hiroshima University, Hiroshima, Japan; M. Sato—Mamoru Sato, National Institute of Agrobiological
Resources, Japan; Y. Takikawa—Yuichi Takikawa, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan. Superscripts: PT, pathotype
strain; T, type strain.
c?: Information was not available.

sition was revealed: in the initial stage, both the catabolic
OCTase gene and the anabolic OCTase gene (argF) existed on the genome of the ancestor of P. syringae; thecatabolic OCTase gene was deleted; the ancestor diverged into the respective pathovars; the argK–tox cluster horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of
only the anabolic OCTase genes (argK and argF) came
to exist on the genomes of these two pathovars.
Incidentally, the research results reported in this paper
have been presented orally (Sawada et al. 1998, 2000a,b,2001).

Materials and Methods
Bacterial Strains
Fifteen strains belonging to 14 P. syringae pathovars, including
pathotype strains, were used (Table 1). The sources of these strains areshown in Table 1, and detailed descriptions of their characteristics are
given in our previous papers (Sawada et al. 1997, 1999). Reference
strains of closely related species, which all belong to Pseudomonas(sensu stricto), were also used for comparison with P. syringae pathovars.

Preparation of High Molecular Weight Genomic DNA
Bacterial cells in the logarithmic phase of growth were suspended in a
10 mM Tris–HCl (pH 7.6) buffer containing 1 M NaCl, adjusted to 109

CFU/mL. Agarose plugs were prepared by mixing equal volumes ofcell suspension and molten 2% low-melting point agarose. Bacterial
cells embedded in agarose were lysed in the presence of RNase, treatedwith proteinase K, and stored in 0.5 M EDTA (pH 9.0) at 4°C. The
agarose plugs containing intact genomic DNA were treated withphenylmethylsulfonyl fluoride (final concentration, 1 mM) to inactivate
proteinase K and washed four times with at least 10 vol of TE [10 mM
Tris–HCl, 1 mM EDTA (pH 8.0)] to remove the phenylmethylsulfonyl
fluoride.

Restriction Digestion and Pulsed-Field Gel
Electrophoresis (PFGE)
It is known that I-CeuI is an endonuclease encoded by a group I intron
in the LSU rRNA gene of Chlamydomonas eugametos, whose cleavage
site exists only within the rrl gene on the genomes of many bacterial
species (Liu et al. 1993). In addition, the number and position of rrl are
strongly conserved among closely related species. Thus, the homologous I-CeuI fragments on different genomes can easily be identified by
combining I-CeuI fragment size comparison using PFGE with Southernblot analysis. This I-CeuI fragment analysis is also advantageous in that
only genomic DNA can be analyzed without being affected by resident
plasmids, because no I-CeuI site exists on a plasmid and because, when
conducting PFGE, closed circular DNA does not permeate the gels but
is trapped in the well (Römling et al. 1996). Thus, I-CeuI fragment
analysis has been employed for analyzing genomes of various bacterial
species (Liu et al. 1993, 1999).
For I-CeuI digestion, agarose plugs were preincubated in a 2×concentrated I-CeuI buffer supplied by the manufacturer for at least 1
h at 4°C and then incubated with 100 ␮L of a fresh 1× buffer containing
10 ␮g of bovine serum albumin (BSA) and 4 U of I-CeuI (New England Biolabs) at 37°C for 4 h to digest DNA in plugs. For partial
digestion with I-CeuI, DNAs in agarose plugs were digested with 1 Uof enzyme in 100 ␮L of a reaction mixture at 37°C, with some variations in the time of digestion. For digestion with all other enzymes,

440
Table 2.

Genetic markers (probea) used for Southern blot analysis and construction of physical maps, and their primersb

Targetgene

Designationof primerb

Sequence ofprimer (5⬘ to 3⬘)

Position insubmitted
sequence

Accession No.

crrs (16S rRNA)

23S-1crrl (23S rRNA)

23S-2drrl (23S rRNA)

rpoD

rpoD

gyrB

gyrB

hrpL

hrpL

hrpS

hrpS

argK

argK

orf3

ORF3e

argF

argF

arcA

arcA

arcB

arcB

arcD

arcD

16BF
16BR-B
23S-F
23S-R
23S-F2
23S-R2
rpo F
rpo R-B
gyr AF3
gyr AR-B3
hrpLF3hrpLR-B
hrpS-F2
hrpS-RB
OCTF-03OCTR
orf3-F
orf3-RargF1FargF1R-B
arcAF
arcAR
arc 1 F 2
arc 1 R-B
arcDF
arcDR

GCTAACTCTGTGCCAGCAGCCGCGG
CGGGACTTAACCCAACATCTCACG
AAGCTTGCTGGAGGTATCAGAAGTG
AAGCTTTTCTTGGAAGCATGGCATC
CGGTGTTTGGCACCTCGATGTCGGC
AAGCCTCACGGGCAATTAGTACTGG
AAGGCGAGATCGAAATCGCCAAGCG
GGAACATGCGCAGGAAGTCGGCTCG
CCGGCGGTAAGTTCGATGATAACTCTGATCGCGGTCAGGCCTTCACGGGC
TTTTGGCTGGCACGGTTATCGCTATA
TGTGGTTTTGCGTGCGAGTTGGTTCC
CTCCAGGCCAAGCTGCTGAGGGTGC
TTGAGCTCGCGGATATTGCCGGGCCGACCGTCAAGGAAGAATTCGGGCGCCGACCTTGTTGACCTCCCG
TGTTGAAGATGTCGGGGTTGTCCGC
TCCACAGGGCGTATCCCGCCCTCGC
CTGCGGTCTGTTGCAAAAAAGGTAG
ATATAGCTATTGCACATGTTGTTGC
TCCACTCCGAAGCCGGCAAACTGCG
TCGCGGTCGCAGAAGCTGAACACGG
TTCCCACAAGGAGATCAATCATGGC
CCTTCGGTGCGGCGATGCGTACGTCTCCAGCCAAAAACTCCGACTAGGAG
ATGTCCAGCTTGAAGGCGAACAGGC

501–525
1094–1071
1216–1240
1569–1545
2476–2500
2887–2863
347–371
928–904
422–446
1090–1066
1025–1050
1677–1652
363–387703–679
835–859
1466–1448
2298–2322
2844–2820
270–294803–779
56–80
893–869
44–68
634–61036–60
565–541

AB001439
AB001439
Y00432
Y00432
Y00432
Y00432
U93292
U93292
X54631
X54631
U16817
U16817
U03852
U03852
M94049
M94049
U27310
U27310M99382
M99382
X14694
X14694
X05637
X05637
M33223
M33223

Designationof probea
16S

Amplicon
size
(bp)
594
354
412
582
669
653
341
632
547
534
838
591
530

a
The products amplified by PCR were cloned, the sequence of the inserts was confirmed, and then the inserts separated by electrophoresis were
excised from the gels and DIG-labeled for use as probes. The conditions of hybridization and detection using the heterologous gene probes in theSouthern blot analyses (Figs. 6 and 7) are described under Materials and Methods and in the legend to Fig. 6.
bAll the primers listed in this table were designed in this study.
c
A probe that hybridizes with the upstream region of the 1-CeuI site.
dA probe that hybridizes with the downstream region of the 1-CeuI site.
e
ORF3 is located in the phtE locus of the argK–tox cluster and is similar to the acetylornithine aminotransferase gene from E. coli (Zhang and Patil
1997).

agarose plugs were immersed in a 1× buffer supplied by the manufacturer and incubated at 4°C for at least 1 h. The buffer was then replacedwith 100 ␮L of a fresh 1× buffer containing 10 ␮g of BSA and 10 U
of enzyme and incubated for 4–16 h. The enzyme-digested DNA fragments were separated by a CHEF-DR II apparatus (Bio-Rad) according
to the manufacturer’s instructions. After PFGE, the gels were stainedwith SYBR Green I (FMC BioProducts). Phage ␭ multimeric DNA andyeast chromosomal DNA were used as molecular mass markers. For
redigestion, restriction fragments separated by PFGE were excisedfrom agarose gels under a long-wavelength UV light, and agarose plugs
containing DNA fragments were trimmed to slices about 1 mm thickand digested with a second enzyme.

Preparation of DNA Probes
For Southern hybridization analysis and construction of physical maps,
13 genes were selected as genetic markers (Table 2). PCR was conducted using the primer sets listed in Table 2, and the genes selected as
markers were amplified. Preparation of templates and amplification by
PCR were performed based on the method described by Sawada et al.
(1995, 1997). After the amplified products were cloned to pUC18, thesequences of cloned inserts were determined, which showed that thetarget amplified products were successfully cloned. The inserts separated by electophoresis were then excised from the gels, labeled with
DIG-High Prime (Roche Diagnostics), and used as probes.

Southern Hybridization Analysis
DNA fragments separated by electrophoresis were transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech). Southernhybridization and detection of the hybridized DNA were carried out
using the DIG DNA Labeling and Detection Kit (Roche Diagnostics)
following the manufacturer’s instructions. In the Southern hybridization analysis conducted for detecting OCTase genes, the following
conditions were established, particularly when heterologous geneprobes were used: hybridization buffer, DIG Easy Hyb (Roche Diagnostics); concentration of the DIG-labeled probe in the hybridization
solution, 25 ng/mL; temperature for hybridization, 40°C; temperature
for washing in 0.5× wash solution, 60°C; and chemiluminescent substrate, CDP-Star (Tropix).

Sequence Alignments
Sequence data were compiled using DNASIS-Mac Version 3.6 (Hitachi
Software Engineering) and MacClade Version 3.06 (Maddison andMaddison 1996), and sequence alignments were determined using
CLUSTAL W version 1.7 (Thompson et al. 1994). DNA sequences for
OCTase-encoding genes were converted into amino acid sequences,and multiple alignments among them were inferred. Some previously
published alignment data (Hatziloukas and Panopoulos 1992; Hough-441
ton et al. 1984; Itoh et al. 1988; Ruepp et al. 1995; Schurr et al. 1995;
Ventura et al. 1997) were used as templates to correct the inferredalignments manually. Information on the functions of gene products
was also used to aid in the multiple alignments. The final products ofthe multiple alignments are available in a machine-readable format.
(For details, contact Hiroyuki Sawada by E-mail at sawada@
affrc.go.jp.) Positions where gaps are present in any of the aligned
sequences were excluded from the following analysis.

gene distribution with the largest width in the codon-usage space. The
kth principal component (PC) is represented by Eq. (2).
M

Z⬘k⳱

兺b X
kj

j

(2)

j⳱1

M

where

兺 b = 1.0.
2

kj

j=1

Phylogenetic Analysis
Phylogenetic trees were constructed using the maximum-likelihood(ML) method (Felsenstein 1981), according to the conditions describedby Sawada et al. (1999). To obtain the topology of ML trees, local
rearrangement searches were conducted using NucML and ProtML of
MOLPHY version 2.3 (Adachi and Hasegawa 1996) with a neighborjoining (NJ) tree topology and a topology obtained by the quick add
OTUs search option as initial trees. The HKY85 model (Hasegawa etal. 1985) was used for the base substitution process, and the “F” optionof the JTT model (Cao et al. 1994) for the amino acid substitution
process. To evaluate the reliability of the inferred ML tree topology, thelocal bootstrap probability (LBP) was calculated using the RELL
method (Hasegawa and Kishino 1994) with 1000 replications.

Analysis of Codon Usage and Adaptiveness of theargK–tox Cluster to the P. syringae Genomes
We characterized the latent structure of the species-specific diversity ofgenes in codon usage for the P. syringae genomes as follows. As wehave described the methodology in one of our previous papers (Kanaya
et al. 1996a), only an outline is given here.
The codon usage pattern for each gene of a species is described bya vector consisting of each codon frequency, i.e., a gene is represented
as a point in the codon frequency space. To describe codon-usage
diversity which was derived from factors such as cellular tRNAamounts and/or mutational pressure for evolutional processes, a scalewith the largest variance in the codon frequency space was estimatedon the basis of principal-component analysis.
Genes which are present on the P. syringae genomes were extracted
from the DDBJ (DNA Data Bank of Japan). We further selected fromthe genes the 33 gene sequences longer than 500 bases in each coding
region that satisfied the following two conditions: (1) genes in which
the sum of codon occurrences for every amino acid except methionine
and tryptophan was not zero were selected; (2) to avoid multiple entriesof one gene, the longest sequence was selected when there were sequences with the same gene name but different coding lengths. In thecase of genes with the same name and length but with variations in theirsequences, one of them was selected arbitrarily. The selected genes
were as follows: argF, argK, avrA, avrB, dapB, hrmA, hrpC, hrpH,
hrpJ, hrpL, hrpR, hrpS, hrpU2, hrpW, hrpX, hrpY, infC, lemA, mrsA,
pgm, pilM, pilP, syrB, syrC, syrD, syrP, tabA, tabB, tblA, tpiA, ttr,
dihydropteroate synthase gene, and fatty acid desaturase gene (DESORF).
To eliminate the influence of gene size, amino acid composition,and codon box number, the codon-usage pattern of the ith gene wasdescribed as a vector xi consisting of xij(m) as expressed by Eq. (1).

冒冋 兺 冒 册M共m兲

xij共m兲 = fij共m兲

fij共m兲 M(m)

(1)

j=1

where fij(m) denotes the jth synonymous codon number of the mthamino acid, and M(m) denotes its codon box number. For simplicity,
variables for individual codon frequencies are denoted Xj. Principalcomponent analysis was conducted to estimate the direction of the

The vector bk ⳱ [bk1, bk2, . . ., bkM] is referred to as the bk vector,
which represents the direction of the species-specific diversity of codon
usage in genes. The kth PC (i.e., Z⬘k) was standardized by Eq. (3) anddenoted Zk.
Zk ⳱ (Z⬘k − Av[Z⬘k])/SD[Z⬘k](3)where Av[Z⬘k] and SD[Z⬘k] are the average and standard deviation of
the variable Z⬘k for the data set. With this scaling, some statistical dataof genes in the PC axes were obtained. For example, if the ith gene hasa zik larger than 2, the codon usage becomes very biased among genesof the respective species because, in statistical theory, only 2.3% ofgenes are included in this interval.
Two parameters, i.e., the proportion in Eq. (4) and the factor loadings in Eq. (5), were used to interpret the PC axes. The former quantifies the width of distribution for the kth PC, and the latter represents
the contribution of the jth variable to the kth PC.

冒兺MPr关Z⬘k兴 = Var关Z⬘k兴

Var关Xj兴(4)

j=1

r共Z⬘k, Xj兲 = Cov关Z⬘k, Xj兴 Ⲑ 共Var关Z⬘k兴Var关Xj兴兲1/2(5)

where Cov[A, B] and Var[A] denote the covariance between variable
A and variable B and the variance of A, respectively.

Results and Discussion
Plasticity of the Genomes of P. syringae Pathovars:
Validation of the Horizontal Transfer of theargK–tox Cluster
An attempt was made to clarify how the genomic structure of the three groups of P. syringae varied while they
evolved separately after diverging from their ancestor. In
addition, “whether or not the argK–tox cluster has actually experienced horizontal transfer” was determined bylocating the argK–tox cluster on the genomes of pv. actinidiae and pv. phaseolicola and by comparing its position between these two pathovars.
Construction of Physical Maps of Genomes by I-CeuI
Fragment Analysis. I-CeuI fragment analysis using
PFGE and Southern blot analysis using the various
probes listed in Table 2 were combined for constructing
physical maps of the genomes of representative strains
selected from the three monophyletic groups which had
been identified by Sawada et al. (1999), i.e., pv. actin-

442

Fig. 1. Results of an I-CeuI partial digestion combined with a Southern blot analysis using a probe that hybridizes with the upstream regionof the I-CeuI site (23S-1; A) and a probe that hybridizes with thedownstream region of the I-CeuI site (23S-2; B). First, the genomic
DNA of P. syringae pv. actinidiae strain KW-11PT (see Table 1) was
partially digested using I-CeuI, then the digests were separated by
PFGE, Southern blotted, and probed (A, 23S-1; B, 23S-2; see Table 2).
In both A and B, the degree of digestion by I-CeuI of the electrophoresed samples gradually advances from Lane 1 to Lane 5, and thesamples in Lane 5 are completely digested. Five I-CeuI fragments
generated by complete digestion are numbered in descending order ofsize (C1 to C5; see Fig. 2). The possible composition of the neighboring I-CeuI fragments, which constitute each partially digested fragment, is indicated in parentheses. Fragments of ca. 1800 kb or smaller
could be resolved satisfactorily by this particular PFGE run (CHEF
DRII, 60 s ramping to 120 s, 200 V, 24 h, and 1.2% gel in 0.5 × TBE
at 14°C), while fragments larger than 2000 kb were resolved by modifying these conditions.

idiae from Group 1, pv. syringae from Group 2, and pv.
phaseolicola from Group 3 (Table 1).
PFGE was conducted for the above three strains on apreliminary basis, and the results showed that nothing
permeated the gels when intact genomic DNA samples
were used, but several fragments were separated whensamples were used after being digested separately witheither I-CeuI, PacI, or PmeI (data not shown). Thisshows that the genome of P. syringae is not linear andthat P. syringae does not contain any linear plasmid.
Subsequently, Southern blot analysis was conducted:
each genomic DNA of the strains tested was first digested separately with either BamHI, EcoRI, EcoRV,
KpnI, PstI, or SalI, which cuts the genomic DNA outside
the rrn genes; then the digests were separated by PFGE,Southern blotted, and probed with either the 16S rRNA
gene (16S, Table 2) or the 23S rRNA gene (23S-2). As
a result, five equally intense bands were detected in allcases (data not shown), showing that five rrn operons
exist on the P. syringae genome.
PFGE was then conducted for the I-CeuI complete
cleavage products, by which five fragments were separated. It was also revealed that the probe that hybridizes
with the upstream region of the I-CeuI site (16S or 23S1; Table 2) and/or the probe that hybridizes with thedownstream region of the I-CeuI site (23S-2; Table 2)

were infallibly hybridized to each of these five fragments(Fig. 1). Furthermore, based on I-CeuI partial digestion
analysis in conjunction with Southern hybridization using the respective probes shown in Table 2 (Fig. 1), these
five I-CeuI fragments were revealed to be linked to eachother on the genome.
These data showed that I-CeuI cleavage sites exist
only within the rrl genes with respect to all three strainstested and that I-CeuI mapping is an effective method foruse in the genomic analysis of P. syringae. It is also
known from these data that the genome of P. syringae iscircular, and five rrn operons exist on it. Hereafter, thefive I-CeuI fragments of the genomes of the respective
strains are referred to as C-1, C-2, C-3, C-4, and C-5, indescending order of size (Figs. 1 and 2).
The I-CeuI partial digestion analysis in conjunction
with the Southern hybridization was conducted in moredetail (Fig. 1), and the order of the five I-CeuI fragments
was obtained as shown in Fig. 2. Incidentally, we had
identified in our previous studies that the order of therespective rRNA genes in the rrn operons of P. syringae
was 5⬘-rrs–rrl–rrf-3⬘ as in various bacterial species(Sawada et al. 1997, 1998, unpublished data). Therefore,
in this study, we succeeded in determining the directionof transcription of each rrn operon (Fig. 2) by using aprobe (16S or 23S-1) that hybridizes with the upstream

443

Fig. 2. Physical maps of the genomes of P. syringae pv. syringae
strain MAFF 302155T (Group 2), pv. actinidiae strain KW-11PT(Group 1), and pv. phaseolicola strain MAFF 302282PT (Group 3).
These maps were constructed and the genetic markers mapped by combining the partial-digestion analyses using I-CeuI and PmeI with theSouthern blot analyses (see Fig. 1 and the text). These genomes are
actually circular but are presented linearly here to facilitate comparisons. I-CeuI fragments are numbered in descending order of size (C1 to
C5), which are shown in the fragment bars. Each junction between the
fragments is the endonuclease cleavage site of I-CeuI. Arrowheads
show the positions and directions of transcription of ribosomal RNA
operons (rrn operons), and the labels above the arrowheads indicate thenames of rrn operons designated by us. The zero point of the genome
map was established in the oriC region, and the five rrn operons werenamed rrnA to rrnE with this zero point as the origin. The genetic
markers are listed in Table 2. Each pair of orthologues is connected byeither dashed or solid lines: the orthologues connected by dashed lines
have positions that are conserved among the genomes, while thoseconnected by solid lines have positions that differ greatly among the
genomes. In the C1 fragment of pv. actinidiae and in the C2 fragmentof pv. phaseolicola, the three genetic markers (argK–tox cluster, hrp
gene cluster, and argF) are placed arbitrarily, so their positions in these
two fragments do not necessarily agree with the actual order of theirpositions on the genomes.

region of the I-CeuI site and a probe (23S-2) that hybridizes with the downstream region of the I-CeuI site
(Fig. 1).
An attempt was also made to identify more precisely
the genomic structure by conducting PFGE analysis using I-CeuI and PmeI. Specifically, genomic DNA
samples were treated in four ways: (1) complete digestion using I-CeuI and complete digestion using PmeI, (2)complete digestion using I-CeuI and partial digestion using PmeI, (3) partial digestion using PmeI of the respective I-CeuI fragments excised from the PFGE gels, and(4) partial digestion using PmeI. The digests of all thesesamples were then separated by PFGE, Southern blotted,
and probed with the respective markers (Table 2) (datanot shown). This helped us to map the marker genes
roughly as shown in Fig. 2. Unfortunately, the arrangement of the argK–tox cluster and the hrp gene cluster in

the C-1 fragment of the pv. actinidiae genome, as well asthat of the argK–tox cluster and argF in the C-2 fragmentof the pv. phaseolicola genome, could not be clearly
determined. For the time being, these two pairs are arranged tentatively in their respective orders as shown in
Fig. 2. More rigorous analysis will be conducted to determine the precise arrangement of the respective genes.
Structure of the P. syringae Genome. The results of
the above analyses showed that all three pathovars tested
had five rrn operons, all of which were present on one
circular genome (Fig. 2). With regard to the genomes ofbacteria, oriC, the origin of replication, is defined loosely
as a locus harboring either the dnaA or the gyrB gene,which is linked to an rrn operon (Cole and Saint Girons
1994). With respect to various bacteria, it has been confirmed that rrn operons are generally arranged in two

444

groups transcribed divergently away from oriC (Coleand Saint Girons 1994; Honeycutt et al. 1993; Le Bourgeois et al. 2000). The genomic structure of P. syringae
obtained in this study showed good agreement with thisgeneral tendency of bacteria. Regarding the genome of
pv. actinidiae, for example, the C-3 fragment is assumed
to contain oriC, and the five rrn operons are arrangedtranscribed toward two opposite directions, going far off
from the C-3 fragment (Fig. 2).
Given that oriC is used as the reference point of bacterial genome maps in general (Römling et al. 1992), thezero point was established in the C-3 fragment of the pv.
actinidiae genome in this study (Fig. 2). With this C-3
fragment as the origin, the five rrn operons were named
rrnA to rrnE in the order shown in Fig. 2. Regarding the
genomes of pv. phaseolicola and pv. syringae, the zero
point was also established in the C-3 fragment, which isassumed to contain oriC, and the respective rrn operons
were named as shown in Fig. 2 with the zero point as theorigin.
Comparison of the Genomic Structure Among theGroups. A comparison of the three strains showed theirgenomes to be very different, not only in basic structure
but also in size. Specifically, the genomes of pv. syringae
and pv. phaseolicola were roughly the same size (about
6 Mb), while the genome of pv. actinidiae, at about 4.7
Mb, was markedly smaller (Fig. 2).
In addition, it was observed that rrn operons andrpoD, which are essential for survival, had undergone
translocation, resulting in a variation of their positions on
the genomes depending on the pathovar (Fig. 2). Specifically, in the case of the pv. syringae and pv. actinidiae genomes, the positions of rrnE and rpoD are relatively close (they coexist in the I-CeuI–PmeI fragment,
which is about 600 kb in size for pv. syringae and about
100 kb for pv. actinidiae) and are arranged in the sameorder in both of these pathovars. On the other hand, rpoDof pv. phaseolicola is present in the C-4 fragment, meaning that its position is opposite the position of rpoD in
the other two pathovars, with the oriC region in the center.
Furthermore, the following results were obtained: (1)Southern hybridization analysis showed that only one
copy of rpoD is present on the pv. phaseolicola genome;(2) a phylogenetic tree of rpoD showed that rpoD of pv.
phaseolicola belongs to the lineage of Group 3 (Sawada
et al. 1999); (3) the results of a t test conducted based on
the distance of synonymous substitution showed that thebase substitution pattern of rpoD of pv. phaseolicola is
not significantly different from that of other essential
genes on the same genome, such as gyrB, hrpL, and hrpS
(Sawada et al. 1999, unpublished data); and (4) the results of PC analysis conducted based on the codon-usage
pattern provided a Z1 value of 0.388 for rpoD of pv.
phaseolicola, indicating that the codon usage of rpoD of

pv. phaseolicola agrees with that of other native genes
existing on the P. syringae genome.
The results of the above four analyses indicate that
rpoD of pv. phaseolicola is not of foreign origin but is anative gene which has resided on the pv. phaseolicola
genome from the beginning. Thus, all these results led us
to speculate that the reason the position of rpoD on the
pv. phaseolicola genome is markedly different from thatof the other two pathovars is because rpoD of pv. phaseolicola translocated from its initial position near rrnE
up to the present position.
Meanwhile, rrnB in pv. phaseolicola was revealed to
be present on the opposite side of that in the other two
pathovars, with the hrp gene cluster in the center, and its
direction of transcription was also opposite (Fig. 2). Another difference revealed was that rrnB and rpoD areclose to each other in the case of pv. phaseolicola (150kb or less), while in the other two pathovars, the closely
located genes are rrnE and rpoD. Based on these results,
rrnB of pv. phaseolicola appears to be an operon orthologous to rrnE of the other two pathovars and to havetransferred to its present position by conducting translocation together with rpoD.
The above results indicate that the structure and sizeof the P. syringae genomes vary greatly depending on
the pathovar, because the P. syringae genomes are quite
rich in plasticity and underwent active and large-scale
rearrangement, including insertion, deletion, and translocation. Similar plasticity has been reported for the genomes of various other bacteria, including Escherichia
coli (Bergthorsson and Ochman 1998; Rode et al. 1999;
Hayashi et al. 2001), Bordetella pertussis (Stibitz andYang 1999), Vibrio cholerae (Khetawat et al. 1999),
Lactococcus lactis (Le Bourgeois et al. 2000), andAzospirillum species (Martin-Didonet et al. 2000). Thismeans in turn that there are diversified genome sizes andstructures even within the same species. The genomes ofE. coli and Salmonella enterica have a size variation of
1 Mb within the same species, respectively (Bergthorsson and Ochman 1998; Hayashi et al. 2001; Rode et al.
1999; Thong et al. 1997). As the difference of about 1.3
Mb in size observed between the genome of pv. actinidiae and the genomes of the other two pathovars is
equivalent to the respective size variations of E. coli and
S. enterica, the plasticity of the P. syringae genomes isconcluded to be quite high.
Given that the genomic structure of pv. phaseolicola
identified in this study is roughly the same as that reported by Esther De Ita et al. (1998), the genomic structure of pv. phaseolicola may be almost invariable. Nonetheless, the structural and size variations within one
single pathovar of P. syringae need to be examined further using more strains with diverse origins, because
there has been a report that the genome size of S. enterica
serovar Typhimurium varies greatly even within this one
serovar (Thong et al. 1997).

445

Phylogenomic Analysis of the argK–tox Cluster: The
argK–tox Cluster Has in Fact Experienced Horizontal
Transfer. The argK–tox cluster was mapped onto thephysical maps constructed as described above, and thentraces which indicate the horizontal transfer of the argK–
tox cluster were searched for by phylogenomically reviewing the phylogenetic data obtained in our previous
studies (Sawada et al. 1997, 1999), as well as the information on the genomic structure obtained in this study.
Except for pv. actinidiae and pv. phaseolicola, no
pathovars of P. syringae have the argK–tox cluster on
their genomes (Figs. 2 and 6) (Sawada et al. 1997, 1998,
1999). Using phylogenetic analysis, we showed in ourprevious paper that these two pathovars belong to two
separate monophyletic groups (pv. actinidiae belongs toGroup 1; pv. phaseolicola, Group 3) and that they arevery distant from each other in a phylogenetic tree
(Sawada et al. 1999). We also proved that the argK sequences of these two pathovars are completely identical,
with not a single synonymous substitution observed(Sawada et al. 1997, 1999). We thus clarified that completely identical argK genes are present on the genomesof these two pathovars, although their genomic structures
have become markedly different from each other (Fig. 2)
as they evolved in two separate courses (Sawada et al.
1999).
In addition, the results of mapping the argK–tox cluster onto the physical maps of genomes showed that theposition of the argK–tox cluster differed greatly between
these two pathovars (Fig. 2); in the case of the pv. actinidiae genome, the argK–tox cluster is in a position
close to the hrp gene cluster, while in the case of the pv.
phaseolicola genome, the argK–tox cluster is present
near argF. Thus, all the above results obtained by thephylogenetic and genomic analyses consistently andstrongly support the hypothesis that the “argK–tox cluster has expanded its distribution onto the genomes ofthese two pathovars by conducting horizontal transfer
after the ancestor of P. syringae diverged into the respective pathovars.”Comparison Between the OCTase Tree and the 16S
rDNA Tree: Validation of the Foreign Origination of
the argK–tox Cluster (1)If the argK–tox cluster has in fact experienced horizontaltransfer, where did it originally come from: pv. actinidiae, pv. phaseolicola, or a bacterial species other thanP. syringae (i.e., of foreign origin)? To resolve this question, various analyses were conducted as discussed here
and in the following sections. In this section, the resultsof a phylogenetic analysis of “argK,” which is a pathogenicity-related gene existing within the argK–tox cluster, are described. This argK is a gene that encodes anabolic ornithine carbamoyltransferase (anabolic OCTase;
EC 2.1.3.3) (Hatziloukas and Panopoulos 1992; Mosqu-eda et al. 1990; Sawada et al. 1997) and is known to havea pathogenicity-related function (Rudolph 1990;
Staskawicz et al. 1980; Templeton et al. 1986).
First, a phylogenetic tree was constructed using thecurrently available published information on OCTase
gene sequences of a variety of organisms to understandthe evolutionary history of OCTase genes, and then thecourse of evolution of argK within this evolutionary history was clarified. Subsequently, another phylogenetic
tree was constructed using 16S rDNA as an index, which
was used to confirm the course of the evolution of the P.
syringae genome. Then these two courses of evolution
were compared to find differences in properties which
could suggest the foreign origination of argK.
Construction of an OCTase Tree: Elucidation of the
Evolutionary History of OCTase Genes. OCTase is
known to consist of two types of enzymes, i.e., anabolicand catabolic, which are involved in the biosynthesis anddegradation of arginine, respectively (Cunin et al. 1986;
Ramos et al. 1967). There are diverse homologues that
encode these two types of OCTase, which are distributed
widely in three domains, namely, Bacteria, Archaea, andEucarya. In this study, such homologues were found tocontain not only orthologues that have originated from
speciation but also paralogues that have originated fromgene duplication (Figs. 3 and 8, Table 6), which is explained later. Thus, to identify the entire aspect of theevolutionary history of OCTase genes, it is essential toconduct phylogenetic analyses precisely and discretelyby sampling homologues (including both orthologues
and paralogues) from a variety of organisms of the threedomains.
The problem, however, is what to use as the outgroupwhen constructing a phylogenetic tree for clarifying theevolutionary history of OCTase genes of all three domains. In this study, we decided to use aspartate carbamoyltransferase (ACTase; EC 2.1.3.2), which is involved in pyrimidine biosynthesis, as the outgroup, andconstructed a composite phylogenetic tree. As for E. coligenomes, it has been reported that pyrB, which encodes
ACTase, is present near 96.4 min, being adjacent to argI,
which is an anabolic OCTase gene (Jacoby 1971). In
addition, it has been pointed out that OCTase and thecatalytic subunit of ACTase are highly homologous withregard to the primary structure and the three-dimensional
structure (Houghton et al. 1984; Van Vliet et al. 1984).
These findings indicate that gene duplication, which occurred prior to the divergence into the three domains,
generated a pair of paralogues, one of which differentiated into the ACTase gene and the other into the OCTase
gene (Cunin et al. 1986; Houghton et al. 1984; Van Vliet
et al. 1984). Thus, a composite phylogenetic tree canwell be constructed using ACTase genes as the outgroup,and the overview of the evolutionary history of OCTase
genes of the three domains can be clarified eventually.
Thus, a maximum-likelihood (ML) tree was con-

446

Fig. 3. OCTase tree with ACTase as the outgroup inferred by the
maximum-likelihood (ML) method based on the JTT-F model. To
clarify the phylogenetic position of argK in the evolutionary history of
OCTase genes, this ML tree was constructed by processing all presently available amino acid sequences of OCTase using the ProtML
program of MOLPHY version 2.3 (Adachi and Hasegawa 1996). The
deepest root was placed arbitrarily at a point between the two lineages
corresponding to the different proteins (OCTase and ACTase). The
horizontal length of each branch is proportional to the estimated number of amino acid substitutions, and the figures above or below theinternal branches show the local bootstrap probability (percentage) obtained for 1000 resamplings. 〫 and ⽧, at the nodes of the tree, show
gene duplication, and 䡬 shows the commencement of speciation ineach domain (lineage). This tree revealed that the ACTase gene and
OCTase gene were generated by gene duplication which took place inthe very early stage of evolution (the node marked by 〫), the OCTase
gene then underwent a second gene duplication (at the node marked by
⽧), and hence two paralogues (categorized into Types A and B here)
now exist in the OCTase gene.

structed as shown in Fig. 3. Gene duplication, which
made genes diverge into ACTase and OCTase genes,
appears at the deepest node of this ML tree (shown by the〫). In addition, it was revealed that gene duplication
occurred one more time in the OCTase lineage (at thenode shown by the ⽧), by which two duplicationoriginated paralogues were generated in the OCTase lineage. It has already been reported that two groups ofprokaryotic OCTase genes exist (Bringel et al. 1997;

Falmagne et al. 1985; O’Reilly and Devine 1994; Ruepp
et al. 1995), and our present phylogenetic analysis
clearly proved that the existence of these two groups is
attributable to gene duplication. In this study, these two
duplication-originated lineages (paralogues) of OCTase
genes are called Type A and Type B (Fig. 3, Table 6).
The Type A lineage contains OCTase genes of only
Bacteria, while the Type B lineage contains OCTase
genes of a variety of organisms belonging to the three447

Fig. 4. Three hypotheses of the evolutionary history of OCTase
genes of all organisms. 䡬 and ⽧, at the nodes of the tree, show
speciation and gene duplication, respectively. A The OCTase tree topology in the case when no OCTase gene duplication occurred in theprocess of evolution. B In the case when OCTase gene duplication
occurred independently only on the genome of Bacteria after the LCA(last common ancestor) diverged into three domains (Bacteria, Archaea, and Eucarya), as a result of which a pair of paralogues, i.e.,Type A and Type B, was generated. C In the case when a pair ofparalogues (Type A and Type B) was generated due to OCTase gene
duplication that occurred on the genome of LCA, followed by divergence into the three domains. The OCTase tree shown in Fig. 3 completely refutes the possibility of the topology in A, and it strongly
supports the topology in C, without totally denying the topology in B.
The branches shown by dashed lines in C denote that OCTase genes
which are assumed to have followed these courses of evolution were
not detected in the samples used in this analysis (Fig. 3).

domains, i.e., Bacteria, Archaea, and Eucarya. However,the bootstrap probability, which determines the branching order of the lineages corresponding to the respective
domains, turned out to be comparatively low (64.8 and85.4%). As the last common ancestor [LCA; the universal ancestor from which all extant life ultimately diverged (Glansdorff 2000)] is suspected to have diverged
into the three domains during a short period of time inview of the time scale of evolution (Miyata et al. 1991),
it may be unlikely to obtain highly reliable results withregard to their branching order using only limited data on
OCTase genes. Thus, no definitive conclusion is given inthis paper with regard to the branching order of the lineages corresponding to the respective domains in the
OCTase tree.
Based on the results obtained above, the evolutionary
history of the OCTase genes of all three domains was
examined. If OCTase genes did not undergo duplication,
their evolutionary history would have been expressed bya simple-shaped topology as shown in Fig. 4A; however,
our analytical results (Fig. 3) refuted this possibility.
Figure 4C shows the evolutionary history of OCTase
genes prepared based on the phylogenetic tree shown in
Fig. 3. Specifically, Fig. 4C shows the following courseof evolution: the OCTase gene underwent duplication
prior to the divergence of LCA; a pair of paralogues,
Types A and B, was generated; and then the divergenceof LCA into the three domains (Bacteria, Archaea, andEucarya) occurred. However, as no definitive conclusions regarding the branching order of the lineages corresponding to the respective domains can be drawn basedon the phylogenetic tree shown in Fig. 3, the topology
shown in Fig. 4B also remains as another possibility,
meaning that the OCTase gene might have duplicatedindependently only in the lineage of Bacteria after LCA
diverged into the three domains. Nonetheless, regardlessof the correctness of either Fig. 4B or Fig. 4C, the basic
course of evolution was clarified as follows: “After the
ACTase gene and OCTase gene were generated by gene
duplication, the OCTase gene was duplicated for a second time; as a result, a pair of duplication-originatedparalogues (OCTase genes, one each belonging to TypeA and Type B, respectively) now exists at least on the
genomes of Bacteria.” These findings are used to examine the foreign origination of argK in the following subsection.
Construction of a 16S rDNA Tree (Criterion for Judging Foreign Origination) and Comparison with the
OCTase Tree. An attempt was made to clarify the“course of evolution of the P. syringae genome” for use
as the criterion for determining the foreign origination of
the argK–tox cluster. Accordingly, another ML tree (Fig.
5), which expresses the phylogenetic relationship of representative Bacteria including P. syringae, was constructed using Archaea as the outgroup and 16S rDNA as
the index. As the branching pattern reproduced in thelineage of Bacteria in this 16S rDNA tree (Fig. 5) agrees
with the widely acknowledged pattern, i.e., “following
the divergence of Thermotoga, the Gram-positive bacteria diverged, then Proteobacteria became further diverged” (Hugenholtz et al. 1998; Pace 1997), this tree is448

Fig. 5. 16S rDNA tree of Bacteria, with Archaea as the outgroup,
obtained by the ML method using NucML (HKY85 model, ␣/␤ ⳱
1.78). The horizontal length of each branch is proportional to the estimated number of nucleotide substitutions, and the figures above or

below the internal branches show the local bootstrap probability (percentage) obtained for 1000 resamplings. This tree shows the phylogenetic relationship among bacterial species which constitute the Type A
lineage of the OCTase tree (Fig. 3).

considered to be an adequate criterion for judging whether or not argK is of foreign origin.
In the 16S rDNA tree, P. syringae belongs to thelineage of the ␥-Proteobacteria and is located very close
to P. aeruginosa (Fig. 5). Incidentally, it has alreadybeen reported that both P. syringae and P. aeruginosa
belong to the genus Pseudomonas (sensu stricto) in the
␥-Proteobacteria and are very closely related to eachother (Anzai et al. 2000; Moore et al. 1996). This isevidently proven by the phylogenetic position of P.
syringae in our 16S rDNA tree shown in Fig. 5.
On the other hand, the Type A lineage in our OCTase
tree shows that argK is in close relation with the OCTase
gene (argF) of Lactobacillus plantarum (a bacterial species belonging to the low-G+C Gram-positive bacteria or
Firmicutes) (Fig. 3). These low-G+C Gram-positive bacteria are known to be extremely distantly related to P.
syringae in terms of evolution (Hugenholtz et al. 1998;
Pace 1997) and are located extremely far from P. syringae in the 16S rDNA tree (Fig. 5). Showing that thecourse of evolution of the P. syringae genome (Fig. 5) isgreatly different from the course of evolution of argK
(Fig. 3), these results strongly suggest the foreign origination of argK.

Species-Specific Diversity of Genes in Codon Usage:
Validation of the Foreign Origination of the argK–tox
Cluster (2)To confirm whether or not the argK–tox cluster is adaptive to the genome of P. syringae, analysis was further
conducted based on the species-specific diversity ofgenes in codon usage using a method developed by Kanaya et al. (1996a, b). This method, which has alreadybeen successfully applied to several bacterial species(Kanaya et al. 1996b, 1997, 1999; Nakayama et al.
1999), constructs a measure based on the widest scale of
the gene distribution in codon frequency space. This axis
is referred to as the first principal component (PC1) andits scale is denoted Z1 (see Materials and Methods).
Analysis of two genes that are present within the argK–
tox cluster, namely, argK and the “fatty acid desaturase
gene” (DES-ORF) (Hatziloukas et al. 1995), provided
the following results.
In P. syringae genomic (native) genes, PC1 accounts
for 30.4% of the total variance, which is much larger thanthose for other PCs (e.g., 10.7% for the second PC). This
indicates that the P. syringae-specific diversity isroughly reflected in PC1. Table 3 shows factor loadings

449
Table 3.

Factor loadings (FL) for the first principal component
NNA codon

Amino
acid
Arg
Leu
Ser
Ala
Gly
Pro
Thr
Val
Ile
Asn
Asp
Cys
Gln
Glu
His
Lys
Phe
Tyr

NNU codon

NNG codon

NNC codon

Codon

FL

Codon

FL

Codon

FL

Codon

FL

CGA
AGA
UUA
CUA
UCA
—
GCA
GGA
CCA
ACA
GUA
AUA
—
—
—
CAA
GAA
—
AAA
—
—

−0.583−0.744−0.785−0.448−0.520—−0.784−0.815−0.415−0.508−0.580−0.717———−0.4570.229—−0.235
—
—

CGU
—
—
CUU
UCU
AGU
GCU
GGU
CCU
ACU
GUU
AUU
AAU
GAU
UGU
—
—
CAU
—
UUU
UAU

0.125
—
—
−0.651−0.740−0.109−0.516−0.121−0.611−0.516−0.718−0.359−0.554−0.760−0.313——−0.364—−0.599−0.172

CGG
AGG
UUG
CUG
UCG
—
GCG
GGG
CCG
ACG
GUG
—
—
—
—
CAG
GAG
—
AAG
—
—

0.317
−0.462−0.431
0.861
0.285
—
0.629
−0.1240.641−0.012
0.667
—
—
—
—
0.457
−0.229
—
0.235
—
—

CGC
—
—
CUC
UCC
AGC
GCC
GGC
CCC
ACC
GUC
AUC
AAC
GAC
UGC
—
—
CAC
—
UUC
UAC

0.743
—
—
−0.183−0.049
0.557
0.602
0.671
0.095
0.597
0.312
0.849
0.554
0.760
−0.313
—
—
0.364
—
0.599
0.172

for PC1 which represent contributions of codon frequencies to PC1. The properties of the codon-usage diversity
revealed in our analysis were as follows: most of thecodon frequencies with A and U at the third codon position contributed negatively to PC1, while most of thosewith C and G at the third codon position contributed
positively to PC1; however, the frequencies for twocodons (GAA and CGU) contributed positively to PC1,
while those for eight codons (AGG, UUG, GGG, ACG,
GAG, CUC, UCC, and UGC) contributed negatively to
PC1.
A t test was conducted to assess the effectiveness of
Z1 as a means of discriminating genomic (native) genes
from foreign genes. Table 4 shows the t values for Z1 andthe GC content at the third codon position (3rd GC),
which were obtained by comparing the average values
and standard deviations of the two groups [the genomic(native) gene group (G-group) and the foreign genegroup (F-group)]. These t values prove that Z1 was better
able to discriminate native genes from foreign genes thanthe 3rd GC, because the significance level of Z1 (p <0.1%) was much lower than that of the 3rd GC (0.1 < p< 1.0%). So Z1 was used to assess the adaptiveness ofargK and DES-ORF to the P. syringae genome, the results of which revealed that the Z1 values of argK (−0.95)
and DES-ORF (−1.36) were very close to the average ofthe foreign gene group (Table 5). This finding could
roughly be confirmed by their 3rd GC values (0.552 and0.554, respectively) as well, which were much lower
than that (0.68) of the G-group (Table 5). Accordingly,
the analytical results obtained by both the codon usage

Table 4. t values between the genomic (native) gene group (Ggroup) and the foreign gene group (F-group)a
G-group

Z1
3rd GCF-group

Av

(SD)

Av

(SD)

t value

0.00
0.68(1.00)(0.10)−1.15
0.57(1.16)(0.14)

4.20
3.36

a
Av and SD represent the group average and standard deviation, respectively. Genes larger than 500 bases in plasmids and in transposable
elements were included in the foreign gene group: avrC, avrD (race 0,
race 1, race 2, race 3, race 6), avrPmaA1, avrPphC, avrPpiB1.R3, cfa3,
cfl, cmaA, cmaT, cmaU, copA, copB, copR, copS, corS, iaaH, iaaL,
iaaM, ptz, rulB, stbA, stbD, tnpA, tnpR, and ethylene-forming enzyme
gene. Genomic (native) genes are listed under Materials and Methods.

and the 3rd GC proved that the argK–tox cluster is offoreign origin.
Thus, all the results obtained by the analyses discussed in this section, the phylogenomic analyses discussed in the previous sections, and the phylogenetic
analyses conducted in our previous studies (Sawada et al.
1997, 1999) strongly and consistently support the hypothesis that the “argK–tox cluster is of foreign origin
and has expanded its distribution by horizontally transferring onto the genome of P. syringae from bacterial
species distantly related to P. syringae.”
Evolution of OCTase Homologues on the P.
syringae Genome
The previous sections have described mainly the resultsof analysis of “argK” existing within the argK–tox clus-

450
Table 5. Comparison between the P. syringae genome and the argK–
tox cluster based on the 3rd GC and codon usage
P. syringae genome

GC content (%)
3rd GC (%)
Codon adaptationc

argK–tox cluster

Whole
genomeaG-groupb

argF

argK

DESORF

56
—
—

56–58
68
—

57.3
67.8
0.00

49.4
55.2
−0.95

49.6
55.4
−1.36

gae genome (Table 5). (iii) All the strains of P. syringae
used in this study were proven to possess the argF gene(Fig. 6). (iv) The position of argF on the genome wasconfirmed to be conserved in all three pathovars investigated (Fig. 2). As these findings showed that the properties of argF are totally opposite those of argK, it can beconcluded unequivocally that “argF is not of foreign
origin but is a native gene that has resided on the P.
syringae genome from the beginning.”aData from Palleroni (1984).
Genomic (native) gene group (see Table 4 and Materials and Methods).
c
Z1 values (see Materials and Methods).
bter. Incidentally, “ArgK,” which is one type of anabolic
OCTase, has been revealed to have a salient pathogenicity-related function as follows: phaseolotoxin, in whosebiosynthesis the argK–tox cluster is involved, is a virulence factor that makes host plants exhibit a disease
symptom (chlorosis) by inhibiting the catalysis of theiranabolic OCTase (Bender et al. 1999); however, phaseolotoxin-producing pathovars (pv. actinidiae and pv. phaseolicola) do not suffer autointoxication because they
possess anabolic OCTase encoded by argK (Hatziloukas
and Panopoulos 1992; Mosqueda et al. 1990; Sawada etal. 1997), which are phaseolotoxin resistant and canfunction even in the presence of phaseolotoxin (Rudolph
1990; Staskawicz et al. 1980; Templeton et al. 1986).
Incidentally, there exists another anabolic OCTase
gene, namely, argF, on the P. syringae genome in addition to argK (Fig. 2). It is known that the isozyme encoded by argF is phaseolotoxin sensitive, is irrelevant topathogenicity, and exerts a housekeeping function when
the argK–tox cluster suspends phaseolotoxin production(Jahn et al. 1985).
Specifically, these OCTase genes (argK and argF) on
the P. syringae genome have important functions that are
essential for the survival of P. syringae. Accordingly, ourinterest focused on the OCTase genes, and they wereanalyzed based on various aspects to clarify the featuresof the OCTase homologues on the P. syringae genome,
as well as the courses of evolution which they have followed.
argF Is a Native Gene. Four findings were obtainedwith regard to argF of P. syringae, as follows. (i) In the
OCTase tree (Fig. 3), argF of P. syringae belongs to theType B lineage, being located extremely close to argF of
P. aeruginosa. This phylogenetic position of argF in the
OCTase tree (Fig. 3) agrees with that of P. syringae inthe 16S rDNA tree (Fig. 5), meaning that, unlike argK,
the course of evolution of argF agrees with that of the P.
syringae genome. (ii) The analytical results obtained bycodon usage and 3rd GC showed that both the Z1 value(0.00) and the value of the 3rd GC (0.678) of argF agreewith the general tendency of other genes on the P. syrin-

Search for the Paralogue of argF (the Orthologue ofarcB of P. aeruginosa): arcB Has Been Deleted from theP. syringae Genome. The finding that argK is foreign andargF is native to the P. syringae genome made us suspect
the existence of one more native OCTase gene on the P.
syringae genome, because the results of our analysis of
OCTase genes proved that paralogues originated fromgene duplication exist in pairs (OCTase genes, one eachbelonging to Types A and B, respectively) on the genomes of Bacteria (Figs. 4B and C, Table 6). Specifically, if the same applies to the P. syringae genome,
there should be an OCTase gene that belongs to Type A,or the paralogue of argF, on the P. syringae genome, as
the other OCTase gene of the pair, argF that belongs toType B, has already been confirmed to exist, as mentioned in the preceding subsection.
Incidentally, P. aeruginosa is known to possess twocopies of OCTase genes on its genome, i.e., a catabolic
OCTase gene (arcB) and an anabolic OCTase gene(argF) (Cunin et al. 1986; Itoh et al. 1988). As with theanalyses discussed in the previous sections, these twocopies of genes were investigated based on codon usage(data not shown), 3rd GC (data not shown), and phylogenetic analysis (Figs. 3 and 5). The results suggested
that both of them are native genes which have resided on
the genome of P. aeruginosa from the beginning. Thismeans in turn that a pair of paralogues, which were generated by gene duplication, has undergone functional differentiation on the genome of P. aeruginosa or its ancestor: one (Type A) into arcB and the other (Type B)
into argF (Fig. 3, Table 6).
With respect to the OCTase genes of P. putida, P.
mendocina, and P. fluorescens, which are extremely
closely related to P. aeruginosa, we deduced that a pairof paralogues (arcB and argF), which have undergone
functional differentiation as with P. aeruginosa, also exist on the genomes of these bacterial species (Table 6)
based on the following three findings: (1) all these bacterial species possess both catabolic and anabolic
OCTases (Cunin et al. 1986; Ramos et al. 1967; Stalon etal. 1984); (2) both of these enzymes can catalyze only aunidirectional reaction (the former catalyzes the phosphorolysis of citruline, and the latter the reverse reaction)(Stalon et al. 1977); and (3) the results of Southern blot
analysis conducted by us, which are discussed later inthis section, showed that both of these bacterial species

451

Fig. 6. Southern blot analysis of the hybridization of OCTaseencoding genes (arcB, argF, and argK) with Pseudomonas species.
The genomic DNAs of Pseudomonas species were digested with either
Aat I (A) or Hind III (B), electrophoresed by the conventional method,
and analyzed by Southern blot hybridization together with all three of
the OCTase gene probes used (arcB, argF, and argK; see Table 2).
Lane 1, P. aeruginosa strain PAO1; Lane 2, P. fluorescens strain
MAFF 302208T; Lane 3, P. putida strain IAM 1236; Lane 4, P. syringae pv. actinidiae strain KW-11PT; Lane 5, P. s. pv. maculicola strain
MAFF 302264PT; Lane 6, P. s. pv. theae strain PT1PT; Lane 7, P. s. pv.
syringae strain MAFF 302155T; Lane 8, P. s. pv. pisi strain MAFF
302269PT; Lane 9, P. s. pv. aptata strain MAFF 302253PT; Lane 10, P.
s. pv. phaseolicola strain MAFF 302282PT; Lane 11, P. s. pv. phaseolicola strain KUZ 1; Lane 12, P. s. pv. glycinea strain MAFF
302260PT; and Lane M, DIG-labeled molecular weight marker (␭DNA
cleaved with HindIII). Conditions of hybridization and detection: hy-

bridization buffer, DIG Easy Hyb; concentration of DIG-labeled probe
in hybridization solution, 25 ng/mL; temperature for hybridization,
40°C; temperature for washing in 0.5 × wash solution, 60°C; andchemiluminescent substrate, CDP-Star. For other conditions for thisSouthern blot analysis, see Materials and Methods. The bands shown
here were detected when a mixture of all three OCTase gene probes(arcB, argF, and argK) was used. It was confirmed in advance that AatIand Hind III had no site in the coding regions of arcA, arcD, or all
OCTase genes of Pseudomonas (sensu stricto) reported up to the present. Accordingly, all native OCTase genes (both Type A and Type B)
and argK existing on the genomes of the respective strains tested weredetected as a single band, respectively. Nevertheless, in both Lane 2 (P.
fluorescens) in A and in Lane 1 (P. aeruginosa) in B, only one band isvisible, because two separate bands, which were actually detected, aresuperimposed at almost the same position.

had two copies of OCTase genes, one each belonging
to Type A and Type B, respectively (Figs. 6A
and B).
Accordingly, it can be expected that a pair of paralogues, which have undergone a similar functional differentiation (Table 6), exists on the genome of P. syringae, because P. syringae is extremely closely related tothese bacterial species belonging to Pseudomonas (sensu
stricto) (Fig. 5). In other words, as one of the pair ofparalogues of P. syringae has been revealed to be an
anabolic OCTase gene (argF) that belongs to Type B, theother paralogue that should belong to Type A is naturally
expected to be a catabolic OCTase gene similar to arcBof P. aeruginosa. Thus, an attempt was made to clarify
the OCTase gene composition on the P. syringae genome
by validating the results obtained by Southern blot analysis.
For the Southern blot analysis, three types of probes
were prepared (Table 2): (1) a probe originated fromarcB of P. aeruginosa [for detecting Type A OCTase
genes from Pseudomonas (sensu stricto)], (2) a probeoriginated from argF of pv. phaseolicola [for detecting
Type B OCTase genes from Pseudomonas (sensu
stricto)], and (3) a probe originated from argK of pv.
phaseolicola [for detecting argK that is foreign to Pseu-

domonas (sensu stricto)]. Preliminary tests of the specificity of the probes (data not shown) showed that theseprobes were capable of detecting genes that are orthologous to the respective genes used as probes from Pseudomonas (sensu stricto) and that they would not hybridize to paralogues under the conditions of hybridizationand detection established for this Southern blot analysis(hybridization buffer, DIG Easy Hyb; concentration of
DIG-labeled probe in hybridization solution, 25 ng/ml;
temperature for hybridization, 40°C; temperature forwashing in 0.5× wash solution, 60°C; and chemiluminescent substrate, CDP-Star).
Five strains were selected from each of the threemonophyletic groups [Groups 1 to 3 (Sawada et al.
1999)] of P. syringae as representative strains, which
numbered 15 strains in all (Table 1). In addition, P. aeruginosa, P. fluorescents, and P. putida, which all belong
to Pseudomonas (sensu stricto), were used as the reference strains. Genomic DNAs prepared from these strains
were digested by using one of either AatI, HindIII, or
NotI [it had been confirmed in advance that these three
restriction enzymes had no cleavage site within the coding regions in any of the genes, such as arcA (for arginine deiminase), arcD (arginine-ornithine antiporter),and all OCTase genes in Pseudomonas (sensu stricto)

452
Table 6. Composition of native OCTase genes presently existing on
the genomes of Pseudomonas (sensu stricto)

Species

Pathovar(s)P. aeruginosa
P. putida
P. fluorescens
P. syringae

actinidiae
phaseolicola
syringae
The otherpathovars

Which OCTase
type?aNo. of native
OCTase genes
presently
existinga

Type
A

Type
B

2
2
2

arcB
(arcB)b(arcB)b

argF(argF)b(argF)b

1c,d
1c,d
1c

—c,d
—c,d
—c

argF
argF
argF

1c

—c

argF

a
OCTase genes of Type A and Type B are a pair of paralogues generated due to gene duplication that occurred in the early stage of evolution (see the text and Figs. 3 and 8). Such native OCTase genes whichcurrently remain on the genome of each species are listed here. Accordingly, those which do not remain on the genomes because they
have been deleted, and those that are “foreign” because they entered the
genomes by conducting horizontal transfer later, are not listed. A —denotes that the gene has been deleted from the genome.
bWith respect to P. putida and P. fluorescens, sufficient data are not
yet available for concluding definitively which type (Types A or B) has
functionally differentiated into which gene [arcB (catabolic OCTasegene) or argF (anabolic OCTase gene)]. However, given that Type B
has differentiated into argF (see the text and Fig. 3) in the case of bothP. aeruginosa and P. syringae, it can be assumed that a pair of paralogues, which have undergone functional differentiation similar to thatof P. aeruginosa and P. syringae, also exists on the genomes of P.
putida and P. fluorescens, because these four species are extremely
closely related to one another. Thus, it appears that their Type A gene
may have differentiated into arcB and their Type B gene into argF.
cThe “native” OCTase gene (presumably a catabolic OCTase gene thatis orthologous to arcB of P. aeruginosa) belonging to Type A of P.
syringae was shown to have been deleted from the genome together
with the arc operon that contained it (see the text and Figs. 6–8).
d“Foreign” argK that belongs to Type A entered the genomes of pv.
actinidiae and pv. phaseolicola by conducting horizontal transfer very
recently (see the text and Figs. 3, 6, and 8).

that have hitherto been reported], then subjected toSouthern blot analysis (Figs. 6 and 7).
Figure 6 shows the results when the mixture of all
three OCTase gene probes (arcB, argF, and argK) wasused at the same time. All the native OCTase genes (bothType A and Type B) and argK existing on the genomesof the respective strains came out as a single band, respectively. However, only one band is visible in Lane 2(P. fluorescens) in Fig. 6A and Lane 1 (P. aeruginosa) in
Fig. 6B, because in these lanes two separate bands,
which were actually detected when each probe was used
separately, are superimposed at almost the same position.
In the case of P. aeruginosa, P. fluorescens, and P.
putida (Lanes 1 to 3 in Figs. 6A and B), which were usedas the reference strains, two separate bands could be
detected from each strain, i.e., one band for each of theorthologues of arcB (Type A) and argF (Type B). Regarding P. syringae, each of the 15 strains tested wasconfirmed to have a single copy of argF (Type B) (Lanes

4 to 12 in Figs. 6A and B). However, in the case of pv.
actinidiae (Lane 4 in Figs. 6A and B) and pv. phaseolicola (Lanes 10 and 11 in Figs. 6A and B), two distinct
bands were detected, which are equivalent to argF (TypeB) and argK (Type A), respectively.
Nevertheless, no band corresponding to an orthologue
of arcB (Type A) of P. aeruginosa could be detectedfrom any of the P. syringae pathovars tested (Lanes 4 to
12 in Figs. 6A and B).
Incidentally, it is known that, in the case of P. aeruginosa, four genes including arcB, which encode the
enzymes of the arginine deiminase pathway (the ADI
pathway), are organized in an operon called thearcDABC operon (Baur et al. 1987, 1989; Gamper et al.
1991). Accordingly, another attempt was made to detect
an orthologue of either arcA or arcD of P. aeruginosafrom P. syringae by conducting a Southern blot analysis(the results shown in Fig. 7 are for arcA only; those for
arcD are not shown); however, no such bands could be
detected from any of the P. syringae pathovars (Lanes 4
to 12 in Figs. 7A and B). Meanwhile, it is also known
that no enzymatic activity of arginine dihydrolase (i.e.,arginine deiminase encoded by the arcA gene; EC
3.5.3.6) can be detected from P. syringae (Palleroni
1984; Sawada et al. 1997). Thus, it can be concluded thatno structure of the arcDABC operon which contains an
orthologue of arcB of P. aeruginosa exists on the genome of P. syringae.
It is known that the arc operon in P. aeruginosa is
involved in the arginine deiminase pathway, which isinducible under anaerobic conditions and serves to generate ATP from arginine (Baur et al. 1987, 1989; Cunin
et al. 1986; Gamper et al. 1991). Nonetheless, it is considered unlikely that P. syringae is placed under suchanaerobic conditions, as it is an epiphyte living mainlyon the plant surface, which is in an extremely aerobic
environment (Hirano and Upper 2000). Accordingly, thearc operon can hardly be an element essential for thesurvival of P. syringae. Thus, it appears that the arc
operon which contained arcB (Type A) was deleted fromthe genome of the ancestor of P. syringae in the phase
before the ancestor diverged into the respective pathovars, and hence only the anabolic OCTase gene (argF)
belonging to Type B remains on the genomes of therespective P. syringae pathovars (Table 6).

Conclusions
P. syringae was shown to be extremely rich in genetic
diversity in our previous studies (Sawada et al. 1997,1999). Specifically, the results of our phylogenetic
analysis of four genes (gyrB, rpoD, hrpL, and hrpS) revealed that the ancestor of P. syringae (the common
ancestor prior to the divergence into pathovars) had so
far diverged into at least three monophyletic groups

453

Fig. 7. Southern blot analysis of the hybridization of the arcA gene with Pseudomonas species. The blot used here is the same as that shown in
Fig. 6. For details of the samples electrophoresed in each lane and the conditions of hybridization and detection, see the legend to
Fig. 6.

(Groups 1 to 3) in the process of its evolution. In addition, the genome of P. syringae was shown to be enormously rich in plasticity in this study. Specifically, thefact that the structure and size of the P. syringae genome
differ greatly among the three groups was confirmed to
be attributable to the active and large-scale genomic rearrangement, including insertion, deletion, and translocation, which occurred while these groups followed different courses of evolution after the ancestor of P.
syringae diverged into the three groups (Figs. 1 and 2).
The argK–tox Cluster Is of Foreign Origin and Has
Expanded Its Distribution by Conducting
Horizontal Transfer
Furthermore, a phylogenomic analysis was conducted inthis study by combining all the information on the molecular evolution, genomic structures, and pathogenicityrelated genes (argK–tox cluster) to clarify the evolutionary mechanism that caused the pathogenic differentiationof P. syringae. The results showed that the argK–tox
cluster has expanded its distribution by transferring horizontally onto the genomes of the two pathovars (pv. actinidiae and pv. phaseolicola) of P. syringae from bacterial species distantly related to P. syringae quite
recently in view of the time scale of evolution (i.e., after
the ancestor of P. syringae diverged into the respective
pathovars).
The argK–tox cluster is involved in the biosynthesisof phaseolotoxin, which is known to be a virulence factor
that causes a disease symptom (chlorosis) by inhibiting
the enzymatic activity of anabolic OCTase in host plants(Bender et al. 1999). Accordingly, these two pathovars(pv. actinidiae and pv. phaseolicola) apparently became
able to cause disease symptoms because they obtained
the argK–tox cluster, which entered their genomes byconducting horizontal transfer, and hence they started

attracting attention as phytopathogenic bacteria. Specifically, the results of this study clarified a dynamic evolutionary mechanism in which the pathogenicity-related
genes (argK–tox cluster), which are present on the genomes, caused a new pathogenic differentiation as a result of conducting horizontal transfer among bacterial
species.
The Evolutionary History of the OCTase Genes
Unfolded on the P. syringae Genome: An Unusual
Composition of OCTase Genes ofPhaseolotoxin-Producing Pathovars
OCTase genes including argK, which are essential forthe survival and pathogenicity of P. syringae (Jahn et al.
1985; Rudolph 1990; Staskawicz et al. 1980; Templeton
et al. 1986), have been the focus of our analyses, reviews,
and deliberations throughout our studies. Using all thedata we have obtained in this and past studies (Sawada etal. 1997, 1999), the “evolutionary history of OCTase
genes unfolded on the genome of P. syringae” was reproduced to scrutinize the relationship between thecourse of evolution of OCTase and the evolution anddiversification of P. syringae. Figure 8 outlines the evolutionary history of OCTase genes.
It was revealed that gene duplication occurred on the
genome of LCA, by which the ACTase gene and OCTase
gene were generated (Fig. 8A), then the OCTase gene
was duplicated one more time, by which a pair of paralogues (OCTase genes belonging to Types A and B)
was generated on the genome of Bacteria (Fig. 8B). It is
likely that such a pair of paralogues subsequently conducted functional differentiation on the genome of thecommon ancestor of Pseudomonas (sensu stricto): one(Type A) into arcB (catabolic OCTase gene) and theother (Type B) into argF (anabolic OCTase gene) (Fig.
8B). Then the arcDABC operon that contained arcB
(catabolic OCTase gene) was deleted from the genome

454

Fig. 8. Evolutionary history of OCTase
genes. A The ACTase gene and OCTase
gene were generated due to gene duplication that took place on the genome of
the LCA (last common ancestor). B After
this, the OCTase gene underwent a second duplication, resulting in the generation of a pair of paralogues (OCTase
genes, one each belonging to Types A
and B, respectively; see Figs. 3 and 4) on
the genome of Bacteria. Furthermore,these paralogues conducted functional
differentiation on the genome of thecommon ancestor of Pseudomonas(sensu stricto): Type A into arcB (catabolic OCTase gene) and Type B into
argF (anabolic OCTase gene) (see Table
6). C Then arcB on the genome of theancestor of P. syringae (the common ancestor prior to the divergence into pathovars) was deleted along with the arc operon that contained it, and only argF
remained on the genome. D Finally, after
the ancestor of P. syringae diverged into
the respective pathovars, the argK–tox
cluster which contained argK (anabolic
OCTase gene) entered the genomes of
pv. actinidiae and pv. phaseolicola frombacterial species other than P. syringae,
creating an unusual gene composition,i.e., “two copies (argK and argF) of only
anabolic OCTase genes exist on the genomes of these two pathovars.”of the ancestor of P. syringae (the common ancestor
prior to the divergence into pathovars), and only argF
remained on the genome (Fig. 8C).
Finally, after the ancestor of P. syringae diverged into
the respective pathovars, the argK–tox cluster containing
argK (anabolic OCTase gene) transferred horizontally
onto the genomes of pv. actinidiae and pv. phaseolicola
from bacterial species distantly related to P. syringae,
and hence an unusual gene composition in which twocopies (argK and argF) of only anabolic OCTase genes
exist on the genomes of these two pathovars was created(Fig. 8D). Having obtained these two anabolic OCTase
genes on their genomes, these two pathovars became
able to select the suitable gene for use at ordinary times(argF) and when they exert phytopathogenicity (argK).
Thus, it was clarified that the genomic rearrangement(duplication, deletion, and insertion) and horizontaltransfer of OCTase genes have played an important rolein the evolution and diversification of P. syringae.

The catabolic gene cluster of xenobiotic-degrading
bacteria and the pathogenicity island of animal pathogens have sometimes been observed to reside on mobile
genetic elements, such as transposons, phages, and conjugative plasmids; thus, the mobile genetic elements are
suspected to have been the cause of the frequent occurrence of genomic rearrangement and horizontal genetransfer, thereby accelerating the evolution and expanding the distribution of the catabolic gene cluster and thepathogenicity island (Hacker and Kaper 1999; Tsuda etal. 1999).
With regard to P. syringae, the in planta transfer ofcoronatine-inducing plasmids has already been identified(Sato 1988). As for pathogenicity-related genes existingon the P. syringae genomes, the results obtained in thisstudy have opened a new horizon to their dynamic evolution as follows. It is quite likely that the large population of P. syringae strains which live saprophytically on
the surfaces of various plants (Hirano and Upper 2000),455

including not only pathogenic but also nonpathogenic
strains, has functioned as the matrix to give birth to new
pathovars (e.g., pv. actinidiae and pv. phaseolicola) by
acquiring pathogenicity-related genes (e.g., the argK–tox
cluster) onto their genomes through horizontal transfer.
Such P. syringae strains may also have functioned as“factories” for creating new pathogenicity-related genes
by generating genetic variations in the process of theirgenomic rearrangement.
Nevertheless, the most important factor that caused
the plasticity of the P. syringae genome, i.e., the molecular mechanism which caused genomic rearrangement
and horizontal gene transfer, still has not been identified.
Thus, in our future studies, we intend to learn when andwhere such a pathogenicity island-like structure was organized, and how it gained the ability to conduct horizontal transfer and expanded distribution, using theargK–tox cluster as a marker. Eventually, we hope to geta more precise understanding of the molecular mechanism involved in the genomic plasticity and diversification of P. syringae.
Acknowledgments. We thank Masami Hasegawa, Yoshifumi Itoh,
Satoshi Oota, Naoko Takezaki, and Yuichi Takikawa for their helpful
suggestions and Junichi Kato, Koushi Nishiyama, Mamoru Sato, andYuichi Takikawa for providing the bacterial strains used in this study.

References
Adachi J, Hasegawa M (1996) MOLPHY; Programs for molecularphylogenetics, version 2.3. Institute of Statistical Mathematics, Tokyo
Anzai Y, Kim H, Park J, Wakabayashi H, Oyaizu H (2000) Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50:1563–1589
Baur H, Stalon V, Falmagne P, Luethi E, Haas D (1987) Primary andquaternary structure of the catabolic ornithine carbamoyltransferase
from Pseudomonas aeruginosa. Eur J Biochem 166:111–117
Baur H, Luethi E, Stalon V, Mercenier A, Haas D (1989) Sequence
analysis and expression of the arginine-deiminase and carbamatekinase genes of Pseudomonas aeruginosa. Eur J Biochem 179:53–
60
Bender CL, Alarcon-Chaidez F, Gross DC (1999) Pseudomonas syringae phytotoxins: Mode of action, regulation, and biosynthesis bypeptide and polyketide synthetases. Microbiol Mol Biol Rev 63:
266–292
Bergthorsson U, Ochman H (1998) Distribution of chromosome lengthvariation in natural isolates of Escherichia coli. Mol Biol Evol
15:6–16
Bradbury JF (1986) Guide to plant pathogenic bacteria. CAB International Mycological Institute, Kew, UK
Bringel F, Frey L, Boivin S, Hubert J-C (1997) Arginine biosynthesis
and regulation in Lactobacillus plantarum: The carA gene and theargCJBDF cluster are divergently transcribed. J Bacteriol 179:
2697–2706
Cao Y, Adachi J, Janke A, Paabo S, Hasegawa M (1994) Phylogenetic
relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on asingle gene. J Mol Evol 39:519–527
Cole ST, Saint-Girons I (1994) Bacterial genomics. FEMS Microbiol
Rev 14:139–160

Cunin R, Glansdorff N, Pierard A, Stalon V (1986) Biosynthesis andmetabolism of arginine in bacteria. Microbiol Rev 50:314–352
Dye DW, Bradbury JF, Goto M, Hayward AC, Lelliott RA, Schroth
MN (1980) International standards for naming pathovars of phytopathogenic bacteria and a list of pathovar names and pathtype
strains. Rev Plant Pathol 59:153–168
Eisen JA (1998) A phylogenomic study of the MutS family of proteins.
Nucleic Acids Res 26:4291–4300
Esther De Ita M, Marsch-Moreno R, Guzman P, Alvarez-Morales A(1998) Physical map of the chromosome of the phytopathogenic
bacterium Pseudomonas syringae pv. phaseolicola. Microbiology
144:493–501
Falmagne P, Portetelle D, Stalon V (1985) Immunological and structural relatedness of catabolic ornithine carbamoyltransferases andthe anabolic enzymes of enterobacteria. J Bacteriol 161:714–719
Felsenstein J (1981) Evolutionary trees from DNA sequences: A maximum likelihood approach. J Mol Evol 17:368–376
Galperin MY, Tatusov RL, Koonin EV (1999) Comparing microbial
genomes: How the gene set determines the lifestyle. In: Charlebois
RL (ed) Organization of the prokaryotic genome. ASM Press,
Washington, DC, pp 91–108
Gamper M, Zimmermann A, Haas D (1991) Anaerobic regulation oftranscription initiation in the arcDABC operon of Pseudomonas
aeruginosa. J Bacteriol 173:4742–4750
Glansdorff N (2000) About the last common ancestor, the universal
life-tree and lateral gene transfer: A reappraisal. Mol Microbiol
38:177–185
Hacker J, Kaper JB (1999) The concept of pathogenicity islands. In:
Kaper JB, Hacker J (eds) Pathogenicity islands and other mobile
virulence elements. ASM Press, Washington, DC, pp 1–11
Hasegawa M, Kishino H (1994) Accuracies of the simple methods for
estimating the bootstrap probability of a maximum likelihood tree.
Mol Biol Evol 11:142–145
Hasegawa M, Kishino H, Yano T (1985) Dating of the human-ape
splitting by a molecular clock of mitochondrial DNA. J Mol Evol
22:160–174
Hatziloukas E, Panopoulos NJ (1992) Origin, structure, and regulationof argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco. J Bacteriol 174:
5895–5909
Hatziloukas E, Panopoulos NJ, Delis S, Prosen DE, Schaad NW (1995)
An open reading frame in the approximately 28-kb tox-argK genecluster encodes a polypeptide with homology to fatty acid desaturases. Gene 166:83–87
Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K, Yokoyama K,
Han C, Ohtsubo E, Nakayama K, Murata T, Tanaka M, Tobe T,
Iida T, Takami H, Honda T, Sasakawa C, Ogasawara N, Yasunaga
T, Kuhara S, Shiba T, Hattori M, Shinagawa H (2001) Complete
genome sequence of enterohemorrhagic Escherichia coli O157:H7
and genomic comparison with a laboratory strain K-12. DNA Res
8:11–22
Hirano SS, Upper CD (2000) Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae—A pathogen, ice nucleus, andepiphyte. Microbiol Mol Biol Rev 64:624–653
Honeycutt RJ, McClelland M, Sobral BWS (1993) Physical map of the
genome of Rhizobium meliloti 1021. J Bacteriol 175:6945–6952
Houghton JE, Bencini DA, O’Donovan GA, Wild JR (1984) Protein
differentiation: A comparison of aspartate transcarbamoylase andornithine transcarbamoylase from Escherichia coli K-12. Proc Natl
Acad Sci USA 81:4864–4868
Hugenholtz P, Goebel BM, Pace NR (1998) Impact of cultureindependent studies on the emerging phylogenetic view of bacterial
diversity. J Bacteriol 180:4765–4774
Itoh Y, Soldati L, Stalon V, Falmagne P, Terawaki Y, Leisinger T,
Haas D (1988) Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: Nucleotide sequence and transcriptional control of the argF structural gene. J Bacteriol 170:2725–2734

456
Jacoby GA (1971) Mapping the gene determining ornithine transcarbamylase and its operator in Escherichia coli. J Bacteriol 108:645–
651
Jahn O, Sauerstein J, Reuter G (1985) Detection of two ornithine carbamoyltransferases in a phaseolotoxin-producing strain of Pseudomonas syringae pv. phaseolicola. J Basic Microbiol 25:543–546
Kanaya S, Kudo Y, Nakamura Y, Ikemura T (1996a) Detection ofgenes in Escherichia coli sequences determined by genome projects
and prediction of protein production levels, based on multivariate
diversity in codon usage. Comput Appl Biosci 12:213–225
Kanaya S, Kudo Y, Suzuki S, Ikemura T (1996b) Systematization of
species-specific diversity of genes in codon usage: Comparison of
the diversity among bacteria and prediction of the protein production levels in cells. In: Akutsu T, Asai K, Hagiya M, Kuhara S,
Miyano S, Nakai K (eds) Genome Informatics Series No. 7. Universal Academy Press, Tokyo, pp 61–71
Kanaya S, Okumura T, Miyauchi M, Fukasawa H, Kudo Y (1997)
Assessment of protein coding sequences in Bacillus subtilis genome using species-specific diversity of genes in codon usage
based on multivariate analysis: Comparison of the diversity between B. subtilis and Escherichia coli. Res Commun Biochem Cell
Mol Biol 1:82–92
Kanaya S, Yamada Y, Kudo Y, Ikemura T (1999) Studies of codon
usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: Gene expression level and speciesspecific diversity of codon usage based on multivariate analysis.
Gene 238:143–155
Khetawat G, Bhadra RK, Nandi S, Das J (1999) Resurgent Vibrio
cholerae O139: rearrangement of cholera toxin genetic elements
and amplification of rm operon. Infect Immun 67:148–154
Le Bourgeois P, Daveran-Mingot, M, Ritzenthaler P (2000) Genome
plasticity among related Lactococcus strains: Identification of genetic events associated with macrorestriction polymorphisms. J
Bacteriol 182:2481–2491
Liu S-L, Hessel A, Sanderson KE (1993) Genomic mapping with I-Ceu
I, an intron-encoded endonuclease specific for genes for ribosomal
RNA, in Salmonella spp., Escherichia coli, and other bacteria. Proc
Natl Acad Sci USA 90:6874–6878
Liu S-L, Schryvers AB, Sanderson KE, Johnston RN (1999) Bacterial
phylogenetic clusters revealed by genome structure. J Bacteriol
181:6747–6755
Maddison WP, Maddison DR (1996) MacClade program and documentation, Version 3.06. Sinauer Associates, Sunderland, MA
Martin-Didonet CCG, Chubatsu LS, Souza EM, Kleina M, Rego FGM,
Rigo LU, Yates MG, Pedrosa FO (2000) Genome structure of thegenus Azospirillum. J Bacteriol 182:4113–4116
Miyata T, Iwabe N, Kuma K, Kawanishi Y, Hasegawa M, Kishino H,
Mukohata Y, Ihara K, Osawa S (1991) Evolution of archaebacteria:
Phylogenetic relationships among archaebacteria, eubacteria, andeukaryotes. In: Osawa S, Honjo T (eds) Evolution of life: Fossils,
molecules, and culture. Springer-Verlag, Tokyo, pp 337–351
Moore ERB, Mau M, Arnscheidt A, Boettger EC, Hutson RA, Collins
MD, Van de Peer Y, De Wachter R, Timmis KN (1996) The determination and comparison of the 16S rRNA gene sequences of
species of the genus Pseudomonas (sensu stricto) and estimation of
the natural intrageneric relationships. Syst Appl Microbiol 19:478–
492
Mosqueda G, Van den Broeck G, Saucedo O, Bailey AM, AlvarezMorales A, Herrera-Estrella L (1990) Isolation and characterizationof the gene from Pseudomonas syringae pv. phaseolicola encoding
the phaseolotoxin-insensitive ornithine carbamoyltransferase. Mol
Gen Genet 222:461–466
Nakayama K, Kanaya S, Ohnishi M, Terawaki Y, Hayashi T (1999)
The complete nucleotide sequence of ␾CTX, a cytotoxinconverting phage of Pseudomonas aeruginosa: Implications forphage evolution and horizontal gene transfer via bacteriophages.
Mol Microbiol 31:399–419
O’Reilly M, Devine KM (1994) Sequence and analysis of the citrulline

biosynthetic operon argC-F from Bacillus subtilis. Microbiology
140:1023–1025
Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740
Palleroni NJ (1984) Genus I. Pseudomonas Migula 1894. In: Krieg NR,
Holt JG (eds) Bergey’s manual of systematic bacteriology, Vol 1.
Williams & Wilkins, Baltimore, pp 141–199
Ramos F, Stalon V, Pierard A, Wiame JM (1967) The specialization of
the two ornithine carbamoyltransferases of Pseudomonas. Biochim
Biophys Acta 139:98–106
Rode CK, Melkerson-Watson LJ, Johnson AT, Bloch CA (1999) Typespecific contributions to chromosome size differences in Escherichia coli. Infect Immun 67:230–236
Römling U, Duchene M, Essar DW, Galloway D, Guidi-Rontani C,
Hill D, Lazdunski A, Miller RV, Schleifer KH, Smith DW, Toschka
HY, Tümmler B (1992) Localization of alg, opr, phn, pho, 4.5S
RNA, 6S RNA, tox, trp, and xcp genes, rm operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO. J Bacteriol 174:327–330
Römling U, Fislage R, Tümmler B (1996) Macrorestriction mapping
and analysis of bacterial genomes. In: Birren B, Lai E (eds) Nonmammalian genomic analysis—A practical guide. Academic Press,
San Diego, pp 165–195
Rudolph K (1990) Toxins as taxonomic features. In: Klement Z, et al.
(eds) Methods in phytobacteriology. Akadimeai Kiado, Budapest,
pp 251–267
Rudolph K (1995) Pseudomonas syringae pathovars. In: Singh US,
Singh RP, Kohmoto K (eds) Pathogenesis and host specificity inplant diseases. Elsevier Science, Oxford, UK, pp 47–138
Ruepp A, Muller HN, Lottspeich F, Soppa J (1995) Catabolic ornithinetranscarbamylase of Halobacterium halobium (salinarium): Purification, characterization, sequence determination, and evolution. J
Bacteriol 177:1129–1136
Sato M (1988) In planta transfer of the genes for virulence between
isolates of Pseudomonas syringae pv. atropurpurea. Ann Phytopathol Soc Jpn 54:20–24
Sawada H, Ieki H, Matsuda I (1995) PCR detection of Ti and Ri
plasmids from phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:828–831
Sawada H, Takeuchi T, Matsuda I (1997) Comparative analysis of
Pseudomonas syringae pv. actinidiae and pv. phaseolicola based onphaseolotoxin-resistant ornithine carbamoyltransferase gene (argK)and 16S-23S rRNA intergenic spacer sequences. Appl Environ Microbiol 63:282–288
Sawada H, Suzuki F, Kanaya S, Matsuda I (1998) Origin and evolutionof argK distributed among Pseudomonas syringae pathovars. In:
Program and Abstracts at the 1998 Annual Meeting of the Phytopathological Society of Japan. Phytopathological Society of Japan,
Tokyo, p 124
Sawada H, Suzuki F, Matsuda I, Saitou N (1999) Phylogenetic analysisof Pseudomonas syringae pathovars suggests the horizontal genetransfer of argK and the evolutionary stability of hrp gene cluster.
J Mol Evol 49:627–644
Sawada H, Kanaya S, Suzuki F, Azegami K, Saitou N (2000a) Horizontal transfer of the argK-tox gene cluster from gram-positivebacteria onto Pseudomonas syringae genomes. In: Proceedings ofInternational Symposium “Evolution 2000,” Annual Meeting of theInternational Society of Molecular Evolution. Center for Information Biology, National Institute of Genetics, Mishima, Japan, pp
44–45
Sawada H, Tsuda M, Suzuki F, Azegami K (2000b) Detection of transposable region from argK-tox cluster of Pseudomonas syringae pv.
phaseolicola. In: Program and Abstracts at the 2000 Annual Meeting of the Phytopathological Society of Japan. PhytopathologicalSociety of Japan, Tokyo, p 119
Sawada H, Kanaya S, Tsuda M, Suzuki F, Tsuchiya K, Saitou N (2001)
Evolutionary history of OCTase genes on Pseudomonas syringae
genomes. In: Proceedings of International Symposium “Evolution-

457
ary Genomics.” Center for Information Biology, National Instituteof Genetics, Mishima, Japan, p 50
Schurr MJ, Vickrey JF, Kumar AP, Campbell AL, Cunin R, Benjamin
RC, Shanley MS, O’Donovan GA (1995) Aspartate transcarbamoylase genes of Pseudomonas putida: Requirement for an inactive
dihydroorotase for assembly into the dodecameric holoenzyme. J
Bacteriol 177:1751–1759
Stalon V, Mercenier A (1984) L-arginine utilization by Pseudomonas
species. J Gen Microbiol 130:69–76
Stalon V, Legrain C, Wiame JM (1977) Anabolic ornithine carbamoyltransferase of Pseudomonas: The bases of its functional specialization. Eur J Biochem 74:319–327
Staskawicz BJ, Panopoulos NJ, Hoogenraad NJ (1980) Phaseolotoxininsensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: Basis for immunity to phaseolotoxin. J Bacteriol 142:720–723
Stibitz S, Yang M (1999) Genomic plasticity in natural populations of
Bordetella pertussis. J Bacteriol 181:5512–5515
Templeton MD, Sullivan PA, Shepherd MG (1986) Phaseolotoxininsensitive L-ornithine transcarbamoylase from Pseudomonas
syringae pv. phaseolicola. Physiol Mol Plant Pathol 29:393–403
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, positions-specific gap penalties andweight matrix choice. Nucleic Acids Res 22:4673–4680

Thong KL, Puthucheary SD, Pang T (1997) Genome size variation
among recent human isolates of Salmonella typhi. Res Microbiol
148:229–235
Tsuda M, Tan HM, Nishi A, Furukawa K (1999) Mobile catabolic
genes in bacteria. J Biosci Bioeng 87:401–410
Van Vliet F, Cunin R, Jacobs A, Piette J, Gigot D, Lauwereys M,
Pierard A, Glansdorff N (1984) Evolutionary divergence of genes
for ornithine and aspartate carbamoyl-transferases—Complete sequence and mode of regulation of the Escherichia coli argF gene;
Comparison of argF with argI and pyrB. Nucleic Acids Res 12:6277–6289
Ventura L, Perez-Gonzalez JA, Ramon D (1997) Cloning and molecular analysis of the Aspergillus terreus arg1 gene coding for an
ornithine carbamoyltransferase. FEMS Microbiol Lett 149:207–
212
Watanabe H, Mori H, Itoh T, Gojobori T (1997) Genome plasticity as
a paradigm of Eubacteria evolution. J Mol Evol 44:S57–S64
Young JM, Takikawa Y, Gardan L, Stead DE (1992) Changing concepts in the taxonomy of plant pathogenic bacteria. Annu Rev
Phytopathol 30:67–105
Zhang YX, Patil SS (1997) The phtE locus in the phaseolotoxin genecluster has ORFs with homologies to genes encoding amino acidtransferases, the AraC family of transcriptional factors, and fatty
acid desaturases. Mol Plant–Microbe Interact 10:947–960