Download Homologous Recombination in Agrobacterium: Potential

Homologous Recombination in Agrobacterium: Potential Implications for the
Genomic Species Concept in Bacteria
Denis Costechareyre, Franck Bertolla, and Xavier Nesme
Université de Lyon, Université Lyon 1, CNRS, and INRA, Ecologie Microbienne UMR 5557, USC 1193, 16 Rue Raphaël Dubois,
Domaine Scientifique de La Doua, Villeurbanne, France
According to current taxonomical rules, a bona fide bacterial species is a genomic species characterized by the genomic
similarity of its members. It has been proposed that the genomic cohesion of such clusters may be related to sexual
isolation, which limits gene flow between too divergent bacteria. Homologous recombination is one of the most
studied mechanisms responsible for this genetic isolation. Previous studies on several bacterial models showed that
recombination frequencies decreased exponentially with increasing DNA sequence divergence. In the present study, we
investigated this relationship in the Agrobacterium tumefaciens species complex, which allowed us to focus on sequence
divergence in the vicinity of the genetic boundaries of genomic species. We observed that the sensitivity of the
recombination frequency to DNA divergence fitted a log-linear function until approximately 10% sequence divergence.
The results clearly revealed that there was no sharp drop in recombination frequencies at the point where the sequence
divergence distribution showed a ‘‘gap’’ delineating genomic species. The ratio of the recombination frequency in
homogamic conditions relative to this frequency in heterogamic conditions, that is, sexual isolation, was found to
decrease from 8 between the most distant strains within a species to 9 between the most closely related species, for
respective increases from 4.3% to 6.4% mismatches in the marker gene chvA. This means that there was only a 1.13-fold
decrease in recombination frequencies for recombination events at both edges of the species border. Hence, from the
findings of this investigation, we conclude that—at least in this taxon—sexual isolation based on homologous recombination is likely not high enough to strongly hamper gene flow between species as compared with gene flow between
distantly related members of the same species. The 70% relative binding ratio cutoff used to define bacterial species is
likely correlated to only minor declines in homologous recombination frequencies. Consequently, the sequence diversity,
as a mechanistic factor for the efficiency of recombination (as assayed in the laboratory), appears to play little role in the
genetic cohesion of bacterial species, and thus, the genomic species definition for prokaryotes is definitively not
reconcilable with the biological species concept for eukaryotes.
Introduction
Even though there is no generally accepted definition
for a bacterial species, according to current taxonomic rules, it is defined by the genomic similarity of its members
(Stackebrandt et al. 2002). Presently, to have an accepted
description of a new species in journals officially in charge
of such publications (e.g., Int J Syst Microbiol Evol), one
must provide results of DNA–DNA hybridization studies
(or accepted alternative methods) to show that species
members globally display a high degree of genome similarity and are clearly different from other known species. According to this strict and formal definition for a bacterial
species, the questions are what are ‘‘genomic species’’
and are bacterial species defined in this way comparable
to some extent to eukaryote species.
A basic and primary difference between prokaryote
and eukaryote species is that prokaryotic species must have
accumulated enough genetic variations through time to be
distinguishable by DNA–DNA hybridization. This implies
that, with genomic species, the speciation events took place
long ago in the past, although nascent species could not be
identified. In addition, contrary to the biological species
concept (BSC) for eukaryotes, the purely operational definition for bacterial species is not supported by any formal biological concept. The BSC, as developed by Mayr
(1942), described that ‘‘species are groups of actually or
potentially interbreeding natural populations, which are reKey words: Agrobacterium, bacterial species, homologous recombination, sexual isolation.
E-mail: [email protected].
Mol. Biol. Evol. 26(1):167–176. 2009
doi:10.1093/molbev/msn236
Advance Access publication October 20, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
productively isolated from other such groups.’’ Considering
genetic exchanges as sexuality, this phenomenon is much
less restricted in bacteria than in eukaryotes, because genes
can be transferred between species. The sexual isolation
concept inherent to the BSC is thus likely not applicable
to prokaryotes.
However, clustering bacteria in discrete groups of
strains on the basis of their genome similarities could be related to sexuality as well. The standard whole-genome
DNA–DNA hybridization technique was the first genomic
method accepted for delineating species (Wayne et al.
1987): ‘‘The phylogenetic definition of a species generally
would include strains with approximately 70% or greater
DNA-DNA relatedness and with 5°C or less DTm. Both values must be considered.’’ With the development of new genetic technology, methods used to define bacterial species are
regularly reevaluated by ad hoc bacterial systematics committees (Stackebrandt et al. 2002; Gevers et al. 2005).
Among the methods accepted for species delineation, amplified fragment length polymorphism (AFLP) clearly highlights that bacterial species are genomically similar and
well-separated clades of strains (Mougel et al. 2002; Portier
et al. 2006). More understandable than the 70% boundary
value of DNA–DNA relatedness or relative binding ratio
(RBR), AFLP revealed that closely related species could
be characterized by maximal intraspecies current genome
mispairing or mismatching (CGM) of 11% and a minimal
interspecies CGM of 15% (Portier et al. 2006). Consequently, Portier et al. (2006) emphasized that there were gaps
in the distribution of genome divergence values, even between closely related species. These gaps currently serve
as the basis for the genomic delineation of bacterial species.
Similarly, a massive comparative analysis based on genome
sequence comparisons of 70 closely related bacteria explored
168 Costechareyre et al.
the genetic boundaries of species (Goris et al. 2007). Goris
et al. (2007) also revealed that the 70% RBR value corresponds to 5% in average nucleotide divergence values for
shared genes. This genetic cohesion of species was also
noted in several multilocus sequence studies (Naser et al.
2005; Thompson et al. 2005; Kuhnert and Korczak 2006;
Richter et al. 2006; Martens et al. 2008). According to this
notion of gaps between species, the pattern of gene diversity
within and between taxonomic units was recently explained
by Martens et al. (2008). These authors calculated, for each
gene and Ensifer species, the amount of ‘‘heterogeneity’’
(highest sequence divergence within a species) and the
amount of ‘‘separability’’ (smallest amount of sequence divergence observed between a particular species and other
species), although the species displaying the smallest amount
of sequence divergence from the particular species is referred
to as the closest neighbor taxon. Indeed, biological mechanisms involved in species delineation could be explored at
the best with particular species displaying large amounts
of heterogeneity and small amounts of separability with their
closest neighbors.
Given these observations, the genetic cohesion of bacterial species is evidently due to a set of ‘‘cohesive forces.’’
It is quite likely that genetic species boundaries may result
from either the existence of ecotypes, corresponding to
ecologically isolated populations, and/or sexual isolation
caused by DNA sequence divergence. Reinforcing this hypothesis, Majewski (2001) reported substantial sexual isolation (i.e., decrease in recombination frequency) of three
orders of magnitude between bacteria showing approximately 10–15% sequence divergence at a marker gene. Accordingly, the 11–15% gap in genome mismatch found by
Portier et al. (2006) suggests that this is the innate parameter
controlling the physiological mechanism of homologous recombination, leading in turn to species formation by way of
efficient genetic isolation. However, until now, sexual isolation reports were only based on genetic divergence without any correlation with whole-genome divergence.
Indeed, several studies have explored the effect of
genomic sequence divergence on the rate of homologous
recombination success in different bacterial models. An
earlier study by Matic et al. (1995) involving an Escherichia coli/Salmonella typhimurium model supported the idea
that ‘‘the genetic barrier that separates these (and perhaps
many other) related species is primarily recombinational.’’
Later studies that explored natural transformation in Bacillus (Roberts and Cohan 1993; Zawadzki et al.1995) or
Streptococcus pneumoniae (Humbert et al. 1995; Majewski
et al. 2000), as well as Hfr conjugation in E. coli/Salmonella
(Vulić et al. 1997), showed that sexual isolation depends,
according to a simple mathematical function, on the level of
DNA sequence divergence in a log-linear mode. This global
relationship was observed at a large scale, but in spite of the
hypothesis that ‘‘gaps’’ in genomic divergence could also
coincide with gaps in sexual isolation, the effects of nucleotide divergence on sexual isolation at species borders have
not been thoroughly examined. Consequently, we address
the question as to whether sexual isolation may or may not
play a direct role in species cohesion, because this isolation
may be measured at the genetic boundaries of genomic species by a significant drop in recombination frequencies. If
this is the case, a conceptual link between the BSC and genomic species definition could be feasible.
To investigate thishypothesis, we comparedvariations in
recombination frequencies with nucleotide sequence divergence in a complex of well-defined genomic species (Popoff
et al. 1984; Mougel et al. 2002; Portier et al. 2006) known as
Agrobacterium biovar 1, but usually and irrelevantly called
‘‘Agrobacterium tumefaciens.’’ This taxon allowed us to extensively explore sexual isolation at the genetic boundaries
of genomic species. Indeed, genomic studies of this taxon
highlighted a broad range of intraspecies divergence within
very closely related but distinct species. In the present study,
a ubiquitous gene, that is, the chromosomal virulence gene
chvA, was sequenced to check whether the clear delineation
ofall known A. tumefaciens genomic species occursatthe gene
scale. Subsequently, the chvA gene, whose sequence divergence was measured, was chosen as the target DNA sequence
for recombination. Thus, we characterized the relationship between recombination frequencies and DNA sequence divergence for very closely related species. Finally, we link the
sensitivity of recombination to DNA sequence divergence
to the occurrence of divergence gaps between species and then
discuss the evolutionary implications of genetic recombination on bacterial speciation.
Materials and Methods
Strains and Plasmids
All strains of the A. tumefaciens species complex used
in this study are listed in table 1. Escherichia coli strains and
plasmids are listed as supplemental data (table 3). Agrobacterium and Sinorhizobium meliloti strains were grown at 28 °C
on YPG medium (yeast extract, 5 g/L; Bacto peptone, 5 g/L;
glucose, 10 g/L). Escherichia coli strains were grown at 37 °C
on Luria Bertani (LB) medium. For recombination experiments, recipient and recombinant strains of A. tumefaciens
were grown on a selective AB mannitol medium (ABM) consisted of AB medium salts of Clark and Maaløe (1967) supplemented with 1 g/l mannitol. Media were complemented with
appropriate antibiotics: 50 lg/ml rifampicin (Rif50), 25 lg/ml
kanamycin (Km25), 25 lg/ml neomycin (Nm25), 50 lg/ml
streptomycin (Sm50), and 100 lg/ml ampicillin (Ap100).
Generation of Rifampicin-Resistant Mutants
Rifampicin-resistant A. tumefaciens strains (C58, TT9,
and M2/1) were obtained by spreading 100 ll of overnight cultures on YPG plates supplemented with 50 lg/ml of rifampicin.
After confirmation of stable rifampicin resistance (RifR), one
clone per strain was arbitrarily selected and stored at 80 °C.
DNA Extraction and Purification
Genomic and plasmid DNA were, respectively, extracted with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) and the QIAprep Spin Miniprep Kit (Qiagen)
according to the manufacturer’s instructions. Polymerase
chain reaction (PCR) products were purified with the GFX
PCR DNA and Gel Band Purification Kit (GE Healthcare,
Little Chalfont, United Kingdom) as recommended by the
manufacturer.
Sexual Isolation of Bacterial Species 169
Table 1
Agrobacterium Strains Used in This Study and Corresponding chvA GenBank Accession Numbers
Reference
GenBank chvA
Accession Number
Crown gall, United States
Dahlia sp., Germany
Plant
Solanum nigrum, root tissue
commensal, LCSA, France
Hybrid poplar, Leuce section, gall,
France
Bulk soil, LCSA, France
Prunus persica x P. amygdalus gall,
cv. GF677, France
De Ley et al. (1973)
De Ley et al. (1973)
Popoff et al. (1984)
EU007383
EU007384
EU007386
Portier et al. (2006)
Portier et al. (2006)
EU007388
Portier et al. (2006)
Portier et al. (2006)
EU007391
EU007389
Genomic species G2
M 2/1
CFBP 5876, LMG 147
CIP 497-74
CFBP 5494, CFBP 2884
CIP 28-75
CFBP 5495
CIP 43-76
CFBP 5496
R
M 2/1 Rif
Ditch water, Belgium
Human blood, France
Human urine, France
Human urine, France
Rifampicin-resistant mutant
Popoff et al.
Popoff et al.
Popoff et al.
Popoff et al.
This study
(1984)
(1984)
(1984)
(1984)
EU007397
EU007398
EU007399
EU007400
Genomic species G3
CIP 107443
CFBP 6623, CIP 493-74
CIP 107442
CFBP 6624, CIP 111-78
Antiseptic flask, France
Human, France
Popoff et al. (1984)
Popoff et al. (1984)
EU007401
EU007402
Apple seedling, Iowa, United States/
A. tumefaciens type strain
Soil around Prunus dulcis, South
Australia
Soil/A. radiobacter type strain
Popoff et al. (1984)
EU007403
De Ley et al. (1973)
EU007406
Popoff et al. (1984)
EU007408
Patient food, France
Human cephalo-rachidian liquid,
France
Popoff et al. (1984)
Popoff et al. (1984)
EU007415
EU007416
Genomic species G6
NCPPB 925
CFBP 5499, LMG 225
Dahlia sp., South Africa
Popoff et al. (1984)
EU007417
Genomic species G7
RV3
CFBP 5500, LMG 317
NCPPB 1641
CFBP 5502, LMG 228
Zutra 3/1
CFBP 6999, LMG 298
No information available
Flacourtia indica, United Kingdom
Malus sp., Israel
Popoff et al. (1984)
Popoff et al. (1984)
Popoff et al. (1984)
EU007419
EU007420
EU007421
Likely hop, United States
Humulus lupulus, gall, Victoria,
Australia
Euonymus alata cv. Compacta, gall,
United States
Rubus macropetalus, OR, United
States
Prunus sp. cv. Montmorency
(cherry), New York, United States
Rifampicin-resistant mutant
Rifampicin-resistant mutant
Popoff et al. (1984)
Popoff et al. (1984)
EU007423
EU007426
Portier et al. (2006)
EU007427
Portier et al. (2006)
EU007428
De Ley (1974)
EU007429
This study
This study
John Innes potting soil, Australia
John Innes potting soil, Australia
Popoff et al. (1984)
Popoff et al. (1984)
EU007430
EU007431
Rhizospheric soil from Prunus
persicae
Portier et al. (2006)
EU007432
S. meliloti 2011 parent strain; SmR
Meade et al. (1982)
AL591793
Strain Name
Other Namesa
Genomic species G1
TT111
CFBP 5767, LMG 196
NCPPB 396
CFBP 5765, LMG 176, Bo542
S56
CFBP 5491, LMG 321
CFBP 5622
CFBP 2517
CFBP 5771
CFBP 2712
Genomic species G4 (Agrobacterium tumefaciens)
B6T
CFBP 2413T, LMG 187T
Kerr 14
CFBP 5761, LMG 15
ATCC 19358
CFBP 5522, LMG 140
Genomic species G5
CIP 107445
CFBP 6625, CIP 291-77
CIP 107444
CFBP 6626, CIP 120-78
Genomic species G8
TT9
CFBP 5504, LMG 64
Mushin 6
CFBP 6550, LMG 201
LMG 75
CFBP 6549
LMG 46
CFBP 6554
C58
CFBP 1903, LMG 287
C58 RifR
TT9 RifR
Genomic species G9
0362
CFBP 5507, LMG 26
0363
CFBP 5506, LMG 27
Genomic species G13
CFBP 6927
Other strain (Sinorhizobium meliloti)
Sm 1021
a
Biological Source, Geographical
Origin, and Other Informationb
CFBP, Collection Francxaise de Bactéries Phytopathogènes, INRA, Angers, France; LMG, Laboratorium voor Microbiologie, Ghent University, Ghent, Belgium; CIP,
Collection de l’Institut Pasteur; ATCC, American Type Culture Collection, Manassas, VA.
b
SmR, Streptomycin resistance.
170 Costechareyre et al.
PCR Amplification and Sequencing
Oligonucleotides used as PCR primers were designed
according to the genome sequence of strain C58 (http://
depts.washington.edu/agro/). The 1,269-bp sequence used
for the phylogenetic study was obtained, depending on the
strains, with either primer pair F2044 (ATT AAC CCT
CAC TAA AGG GAT TCG GCC GWA TCA TYG
ACG C) and F2047 (TAA TAC GAC TCA CTA TAG
GGC GAT GAT GAA GGT CGT CC) or primer pair
F2880 (CCG TTG TCG TCA TCG CCA) and F2883
(AAC AGG ACG AGA TCG GCA TC), amplifying
1,457 or 1,558 bp, respectively. The 1,401-bp fragments
used to clone chvA alleles in pK18mob were obtained with
primers F2650 (GGC GGG YTT CGG CGT CT) and
F2651 (CGG TGR GCG ATG ATG AAG GT) designed
to amplify chvA in Agrobacterium strains as well as the homologous ndvA in S. meliloti. PCRs were performed using
Taq polymerase (Invitrogen, Carlsbad, CA) with initial denaturation at 94 °C for 4 min, followed by 34 cycles with
denaturation at 94 °C for 1 min, annealing at a strainadjusted temperature (54–62 °C) for 1 min, and extension
at 72 °C for 1 min 20 s. Purified PCR products were sequenced (Genoscreen, Lille, France) with primers already
used for amplification, except for F2044 and F2047, which
have T3 and T7 tails at the 5#-terminus, respectively.
Construction of Plasmids for Homologous
Recombination
chvA PCR products were cloned into the pCR2.1TOPO cloning vector, and then, plasmids were used to
transform E. coli DH5a competent cells, according to the
manufacturer’s instructions (Invitrogen, Paisley, United
Kingdom). Appropriate restriction endonucleases allowed
us to select plasmids that contained inserts in the same direction, and then, the sequences of all cloned PCR products
were verified using standard M13 forward and M13 reverse
primers. Fragments carrying chvA alleles were released
with BamHI and XbaI and cloned into a pK18mob vector
previously digested with the same enzymes. This generated
14 different pK18-chvA mobilizable vectors that were used
to transform competent E. coli S17-1 cells (Simon et al.
1983) using the standard protocol (Sambrook and Russell
2001). Because the tra gene required for pK18mob transfer
was present in the chromosome of S17-1, transformations
were performed by biparental matings. Transformant cells
were selected on LB Km25 Nm25 Ap100 medium.
Sequence Analysis
Sequence data were manually edited and automatically assembled using Lasergene’s DNAStar SeqMan program (version 7.1). Multiple alignments and determination
of the percentage nucleic acid identity were performed using Lasergene’s DNAStar Megalign ClustalW alignment
(version 7.1). Phylogenetic trees were constructed using
ClustalX software (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) to obtain a bootstrap neighbor-joining tree (1,000
bootstrap data replicates). DnaSP software (version 4.1)
(Rozas and Rozas 1999) was used to determine the G þ
C content. The BlastN program (Altschul et al. 1990) on
the NCBI Web site was used to compare sequences of plasmid pK18mob and chvA alleles with the Agrobacterium
strain C58 genome sequence.
Measurement of Recombination Frequencies by the
Bacterial Mating System
The pK18mob plasmid self-replicates in E. coli, but its
maintenance in Agrobacterium is only possible by integrative recombination (Simon et al. 1983; Schäfer et al. 1994).
Homologous recombination frequencies, depending only
on chvA sequence divergence, were determined using a biparental conjugation system involving E. coli S17-1 (pK18chvA) donor cells and Agrobacterium RifR recipient cells. A
single Agrobacterium RifR colony was inoculated into 7 ml
of YPG Rif50 and grown at 28 °C until the exponential
growth phase at OD600 5 0.8 (approximately 18 h). For
E. coli S17-1 (pK18-chvA), a single colony was inoculated
into 2.5 ml of LB Km25 Nm25 for overnight growth at
37 °C. Fifty microliters of the preculture was then used
to inoculate 4 ml of LB Km25 Nm25 before incubation at
37 °C until OD600 5 0.8 (approximately 5 h). A 1:10 donor:recipient ratio was used for bacterial mating, which corresponds to 108 E. coli S17-1 (pK18-chvA) mixed with 109
Agrobacterium RifR cells. Appropriate volumes of donor
and recipient cell cultures were separately washed twice
and resuspended in 50 ll of a cold salt solution (NaCl
0.9 g/L). Donor and recipient cells were mixed and incubated at room temperature for 5 min before spotting totally
100 ll on a 0.2-lm Isopore membrane-filter (Millipore)
placed on an LB plate. Conjugation mixtures were incubated for 16 h at 28 °C, and then, cells were resuspended
in 4 ml of MgSO4 10 mM. Appropriate dilutions in
MgSO4 10 mM were plated on selective medium. Escherichia coli donor cells were counted on selective LB Km25
Nm25 medium and incubated for 24 h at 37 °C. Agrobacterium recipient cells were counted on selective ABM
Rif50 medium after approximately 72-h incubation at
28 °C. Agrobacterium recombinants were counted on selective ABM Rif50 Km25 Nm25 medium while counterselecting donor and nonrecombinant recipient cells, after
approximately 72 h at 28 °C. The recombination frequency
was calculated as the number of recombinant cells divided by
the number of donor cells. The sexual isolation ratio was determined by dividing the homogamic (i.e., perfect match) recombination frequency by the heterogamic frequency for
each trial and each donor recipient. At least four independent
crosses were done per mating pair and then averaged.
As a control, conjugations between E. coli S17-1 harboring an empty pK18mob and Agrobacterium RifR strains
were performed independently in quadruplicate, and no
Agrobacterium recombinant cells were obtained.
Molecular Screening of Recombinants by PCR and
Sequencing
The presence of pK18-chvA inserts in the recipient
chvA gene was verified by PCR and sequencing for at least
one clone of each kind of recombinant. To check for the
presence of chvA tandem sequences resulting from
Sexual Isolation of Bacterial Species 171
FIG. 1.—Neighbor-joining tree based on chvA sequences (1,299 bp) of Agrobacterium strains. The homologous ndvA gene from Sinorhizobium
meliloti 1021 was used as the out-group. The three strains used as recipient cells are indicated by arrows. Strains corresponding to the chvA donor are
underlined. Bootstrap % values (70%) obtained from 1,000 data resamplings are shown.
integrative recombination, primers F2044 and F5039 (TAA
CCG TAT TAC CGC CTT TGA G) were used to amplify
the 1,990-bp ‘‘upstream hybrid fragment’’ and primers
F2785 (TAA GTT GGG TAA CGC CAG GGT T) and
F2787 (GGA ACA GGA CGA GAT CGG CAT) were used
to amplify the 1,578-bp ‘‘downstream hybrid fragment’’
(supplemental data, fig. 5). PCR products of hybrid fragments were sequenced as described above.
Statistical Analysis
Linear regression and covariance analyses were performed using RÓ software (http://www.r-project.org/).
Nucleotide Sequence Accession Numbers
The sequences have been deposited in GenBank, and
the accession numbers are listed in table 1.
Results and Discussion
Sequence Analysis of chvA
The present work was undertaken to evaluate the degree of sexual isolation at genomic species boundaries. For
this purpose, chvA was chosen because it is both: 1) not
essential for growth and 2) essential for ecological functions in relation to plant interactions (Breedveld and Miller
1994). This gene can therefore be both disrupted for use in
recombination studies and found in all agrobacteria for nucleotidic sequence analysis.
The nucleotide sequence of chvA was determined for
a large set of strains belonging to all known species of the
A. tumefaciens complex (table 1). chvA was found to be useful
for the delineation of all genomic species (fig. 1) and to have
a G þ C content ranging from 59.1% to 61.6%, which is close
to the 59.4% G þ C content of the C58 circular chromosome
(data not shown). In addition, chvA appeared to be highly correlated with current genome divergence values (CGM), with
a broad range of genetic divergences between pairs of strains
evenly distributed close to the within-to-between species
boundaries (fig. 2). However, although the CGM cutoff for
A. tumefaciens species was found to be 15% (Portier et al.
2006), it was found to be 4.5% for chvA in the present study,
that is, a value remarkably close to the 5% average nucleotide
mismatch cutoff defined by Goris et al. (2007) to delineate
bacterial species. Finally, the chvA phylogeny is congruent
to those obtained with a variety of other genes (recA, mutS,
gyrB, glgC, and gltD; data not shown), indicating that chvA
is not particularly prone to recombination between or within
species. chvA is thus a relevant alternative for usual whole-genome comparisons carried out for species assignation, and it
could also beused to testfinemodificationsin thesensitivity of
homologous recombination to gene divergence at boundaries
of bacterial species.
Sensitivity of Recombination to chvA Sequence
Divergence
The recombination frequency is indeed variable from
one gene to another, ranging for instance from two to more
172 Costechareyre et al.
ilar recombination frequencies with the two G8 recipient
strains C58 and TT9 and a lower level with the G2 recipient
strain M2/1 (supplemental data, fig. 6). This individual
strain effect was eliminated after data transformation into
sexual isolation values (table 2). As described by Roberts
and Cohan (1993), the relationship between sexual isolation
and sequence divergence (fig. 3) is given by a log-linear
function:
log10 ðqÞ 5 /p þ a
FIG. 2.—Correlation between chvA sequence mismatch and CGM
values in Agrobacterium tumefaciens: ‘‘gaps’’ in their distribution,
delineating genomic species, are shaded. CGM values were determined
by AFLP (Portier et al. 2006), and sequence mismatches were determined
in this study. The graph was drawn by combining data from 225 pairwise
comparisons.
than four orders of magnitude among genes of the same genome in Ralstonia solanacearum (Fall et al. 2007). In the
present study, we showed that chvA recombined up to 102
in homologous conditions. This value is high and comparable to the level noted with other marker genes in previous
studies (Roberts and Cohan 1993; Humbert et al. 1995; Zawadzki et al. 1995; Majewski and Cohan 1998; Majewski
et al. 2000). As it is easier and more accurate to compare
data obtained with genes displaying high recombination
frequencies, chvA thus appeared to be a suitable marker
gene for studying recombinations.
The homologous recombination sensitivity to divergence of chvA was tested relative to genomic species G8
and G2 by using C58 and TT9, and M2/1, respectively,
as recipient strains, mated with clones of chvA alleles from
a set of strains (fig. 1) displaying wide divergence with respect to recipient strains (table 2). The results showed sim-
with q being the sexual isolation, / the sensitivity of sexual
isolation to sequence divergence p, and a the y-intercept.
No significant differences were found between slopes
(F3.17 5 50.22, P 5 0.39 for /C58 and /TT9; F3,16 5
32.34, P 5 0.97 for /C58 and /M2/1; F3.11 5 23.62,
P 5 0.57 for /TT9 and /M2/1). The average sensitivity
of sexual isolation to sequence divergence in Agrobacterium is / 5 11.58. No significant differences in y-intercept
values were found between aC58 and aM2/1 (F2.17 5 51.54,
P 5 0.75) and between aTT9 and aM2/1 (F2.12 5 37.29, P 5
0.06). There was a significant difference in the y-intercept
value between strains aC58 and aTT9 (F2.18 5 75.85, P 5
0.003).
Departure from Exact Log-Linear Regression
Paradoxically, the y-intercept in figure 3 is significantly different from zero (P . 0.05), suggesting that sexual isolation could occur even in homogamic conditions.
Careful inspection of the curves revealed that the log-linear
regression actually fitted better for nucleotide divergence
ranging from 1% to 10%. Actually, with the three recipient
strains, a dramatic increase in sexual isolation ratios was
observed when comparing homologous conditions with heterologous crosses involving the most closely related foreign
alleles (i.e., an increase of 4.3 for 1.2% divergence, 3.2 for
Table 2
chvA Sequence Divergence, Recombination Frequencies, and Sexual Isolation Measured for Each Donor/Recipient chvA
Allele in Agrobacterium tumefaciens
Recipient Strains
Donor chvA
Allele
C58
LMG 75
LMG 46
Mushin 6
TT9
NCPPB 925
RV3
B6
Kerr14
M2/1
CFBP 6927
CIP 497-74
CIP 107442
Sm 1021 (ndvA)d
a
C58
Divergence
0
0.012
0.017
0.036
0.043
0.064
0.086
0.092
0.093
0.101
0.101
0.103
0.103
0.243
TT9
RFa
SIb
Divergence
c
2.00 ± 0.05 0
2.72 ± 0.16 0.63 ±
2.72 ± 0.18 0.70 ±
2.76 ± 0.08 0.83 ±
2.87 ± 0.08 0.91 ±
2.93 ± 0.12 0.96 ±
3.18 ± 0.06 1.21 ±
3.30 ± 0.08 1.29 ±
3.30 ± 0.08 1.33 ±
3.32 ± 0.02 1.39 ±
3.58 ± 0.10 1.61 ±
3.68 ± 0.06 1.71 ±
3.78 ± 0.07 1.81 ±
No transformants
0.07
0.09
0.05
0.04
0.08
0.03
0.06
0.05
0.02
0.06
0.03
0.04
0.043
0.036
0.031
0.012
0
0.071
0.081
0.087
0.09
0.101
0.105
0.105
0.097
0.238
RFa
M2/1
SIb
2.91 ± 0.04 0.65 ±
NT
NT
2.71 ± 0.05 0.50 ±
2.23 ± 0.02 0c
3.01 ± 0.09 0.78 ±
NT
NT
3.35 ± 0.08 1.08 ±
NT
3.72 ± 0.11 1.47 ±
3.40 ± 0.05 1.18 ±
3.30 ± 0.07 1.11 ±
No transformants
Divergence
0.03
0.03
0.06
0.08
0.10
0.03
0.05
0.101
0.10
0.098
0.101
0.101
0.102
0.084
0.089
0.091
0
0.103
0.009
0.094
0.246
RFa
SIb
3.64 ± 0.10 1.42 ±
NT
NT
NT
NT
3.68 ± 0.09 1.46 ±
3.43 ± 0.10 1.17 ±
NT
NT
2.24 ± 0.12 0c
4.11 ± 0.04 1.90 ±
3.09 ± 0.07 0.75 ±
3.61 ± 0.09 1.25 ±
No transformants
0.02
0.05
0.04
0.02
0.06
0.05
RF 5 Log10(recombination frequency) ± standard error, based on at least four independent experimental assays. NT 5 not tested.
SI 5 Log10(sexual isolation ratio) ± standard error. The values are obtained by dividing the frequency of homologous condition by the frequency of heterologous
condition for each trial for each combination tested and then averaged.
c
The sexual isolation ratio in homologous conditions (between a recipient strain and its own chvA allele) is set at zero.
d
No transformants detected with the use of the ndvA gene, corresponding to a recombination frequency lower than the experimental detection limit, 2 106.
b
Sexual Isolation of Bacterial Species 173
This length matches MEPS length values reported in the
literature: 23–50 bp for E. coli (Shen and Huang 1986;
Smith 1988) or inferred by us from reported / estimates,
that is, 49 bp for Bacillus subtilis and 42 bp for St. pneumoniae (Majewski and Cohan 1998; Majewski et al. 2000).
Although A. tumefaciens (C58) is not known to be naturally
competent, the homologous recombination system is efficient, as in other taxa known to be naturally competent,
such as Bacillus and St. pneumoniae.
Conclusions
FIG. 3.—Log10(sexual isolation) versus chvA sequence divergence.
Results for the three Agrobacterium recipient strains are represented, C58
(d), TT9 ()), and M2/1 (D). The average value of the three slopes
/Agrobacterium 5 11.58 corresponds to the average sensitivity of sexual
isolation to DNA sequence divergence in Agrobacterium species.
1.2%, and 5.6 for 0.9%, between C58 LMG 75, TT9 Mushin 6, and M2/1 CIP 497-74, respectively). Conversely, another dramatic increase was observed with matings
involving alleles with more than 10% divergence (fig. 3).
Molecular Analysis of chvA Recombinant Strains
No significant sequence identity of chvA alleles or
pK18mob was detected by the BlastN program within
the whole C58 genome sequence (except chvA). All Km,
Nm, and Rif resistant colonies later obtained with pK18mobchvA were thus primarily assumed to be bona fide recombinant strains resulting from single homologous recombination in the target chvA gene. This was experimentally
verified by sequencing the insertion zone of at least
one putative recombinant colony per cross. The sequences
unambiguously showed that all tested recombinants had
integrated pK18mob-chvA into the recipient chvA gene
(data not shown).
Efficiency of the Homologous Recombination System in
A. tumefaciens
According to previous studies in E. coli (Rayssiguier
et al. 1989; Matic et al. 1995; Vulić et al. 1997; Štambuk
and Radman 1998; Vulić et al. 1999), Bacillus (Zawadzki
et al. 1995; Majewski and Cohan 1998, 1999), and St. pneumoniae (Humbert et al. 1995; Majewski et al. 2000), the
two major factors limiting recombination are the mismatch
repair system and the difficulty in heteroduplex formation,
mediated by the RecA protein. To initiate recombination,
RecA requires a minimum stretch of consecutive base pairs
of perfect identity or minimal efficient processing segment
(MEPS) between the recipient and donor (Shen and Huang
1986). The MEPS length differs among bacteria due to RecA diversity. As a result, in the present study, the MEPS
length (n) was estimated at ;27 bp in Agrobacterium by
using the formula of Majewski and Cohan (1998):
n 5 / ln 10:
Majewski (2001) indicated that ‘‘bacterial species experience a degree of sexual isolation from genetically divergent organisms since recombination occurs more frequently
within species than between species.’’ Data reported in this
review enabled us to speculate that bacterial species are
highly and significantly sexually isolated, because 9% divergence at the marker gene between Haemophilus, Pseudomonas, or Streptococcus results in a drop of three orders
of magnitude in recombination efficiency. Nevertheless, the
drop in recombination efficiency, that is, the sexual isolation, was only 3-fold between B. subtilis and Bacillus mojavensis displaying only 4.6% divergence at the marker
gene, indicating that these two Bacillus species are closely
related. We thus assessed the reliability of this result in
other taxa by thoroughly examining sexual isolation in
the A. tumefaciens genomic species complex.
Genomic species are defined by a ‘‘gap’’ in the wholegenome divergence distribution, and such gaps also occur
in the distribution of most genes such as chvA, so the multiple loci sequence analysis method is efficient for taxonomy (Gevers et al. 2005). However, there is no such gap
or drop in the distribution of sexual isolation values in
the G8 model species (fig. 4), likely because the most proximal out-strain, that is, NCPPB 925 from G6, is very closely
related to G8. Hence, at the within-to-between species border, for approximately 4.3–6.4% nucleotide divergence,
sexual isolations were 8 and 9, respectively. In other words,
with the chvA model, there was only a 1.13-fold decrease
(i.e., 9/8) in recombination frequency for a nucleotide divergence difference of 2.1% (i.e., 6.4–4.3), thus for a narrow
border between species (i.e., maximal intraspecies divergence within the heterogeneous genomic species G8
compared with minimal interspecies divergence between
closely related G8 and G6). Indeed, the nucleotide divergence differences at species borders vary according to genes
and species. Nevertheless, multilocus sequence analysis
(MLSA) performed with A. tumefaciens showed that, except in cases of interspecies gene transfers, the smallest nucleotide divergence gaps at species borders of G8 were
2.3%, 3.3%, 5.4%, 5.0%, and 3.6% for recA, gyrB, mutS,
gltD, and glgC, respectively (D. Costechareyre, F. Bertolla
and X. Nesme, unpublished data). Assuming that the average sensitivity of sexual isolation to sequence divergence in
Agrobacterium (/ 5 11.58) is the same for all genes, it is
thus likely that the drop in recombination frequency between closely related but highly heterogeneous species varied from 1.4- to 4.2-fold at most. From a physiologic
standpoint as determined in the laboratory and for a single
174 Costechareyre et al.
FIG. 4.—Graph representing the nucleotide sequence divergence and
the sexual isolation ratio between the reference strain C58 and other
related strains. The DNA sequence divergence determined by AFLP (*)
or with chvA sequences (), and the sexual isolation ratio (d) are
represented.
cross-over, declines in recombination frequency from
within-to-between species are not different enough to significantly hamper gene flow between closely related species
as compared with gene flow between distantly related
strains within a species. Nevertheless, sexual isolation of
a G8 member (C58) from the other nondistantly related genomic species of the A. tumefaciens complex varied gradually from 16 to 63 for G7 and G5, respectively, thus
decreasing the chance of gene flow accordingly. Comparatively, we found a much more substantial drop in recombination efficiency (4.3-fold) with the least
mismatching allele displaying 1.2% nucleotide divergence
(fig. 3). For recombination involving different strains within
the same species instead of different members of the same
clone, sexual isolation actually occurs, thus contributing to
preserving the genome identities of each clone or strain.
A consequence of the genomic species definition for
bacteria is that the same genome divergence cutoff is used
regardless of the class. It spans around 5% of average nucleotide mismatch values for shared genes as determined
with complete genome sequences (Goris et al. 2007). More
precisely, MLSA showed that nucleotide divergence patterns within and between species for various genes are very
similar in specific comparable genera. For instance, the
maximal divergence within Vibrio cholerae was 5.2%
for recA, and the minimal interspecies divergence of V.
cholerae with its closest neighbor Vibrio mimicus was
7.3% (Thompson et al. 2005) dealing with a minimal nucleotide divergence gap of 2.1%. For the same gene but in
the Burkholderia cenocepacia complex, the minimal gap
between genomovar III-B and III-C was 1.9% (i.e., 4.5–
2.6; Baldwin et al. 2005). Comparably, in Ensifer/Sinorhizobium spp. (formerly Sinorhizobium spp.), the maximal
heterogeneity within species is 1.2%, 1.3%, and 2.8%
for dnaK, recA, and gyrB, for Ensifer arboris, Ensifer saheli, and Ensifer fredii, respectively. The respective minimal interspecies divergence of these genes for these species
was 4.2%, 7.3%, and 6.7%, with Ensifer meliloti, Ensifer
kostiensis, and Sinorhizobium americanum, respectively
(Martens et al. 2008), yielding 3%, 6%, and 3.9% minimal
divergence gaps, respectively, in this genus. Of course, with
less heterogeneous or less related species, divergence gap
values can be larger, for example, reaching 2% and 11% for
rpoA and pheS, respectively, in Enterococcus spp. (Naser
et al. 2005). Nevertheless, as a result, very similar divergence gap values have been found between closely related
but heterogeneous species with several genes in several
other genera. This suggests a similar minor effect of homologous recombination at species borders in other taxa. However, as the sensitivity of sexual isolation to sequence
divergence is generally not known in other genera, it is presently not possible to quantify the corresponding declines in
sexual isolation.
In conclusion, the present definition of a bacterial
species is based on the occurrence of a maximal genome
divergence threshold that was empirically found to be almost the same in many taxa (Wayne et al. 1987). The delineation of genomic species in the A. tumefaciens species
complex perfectly fits this definition with roughly the same
canonical 70% RBR threshold value (Popoff et al. 1984).
The present study of patterns that occur at species borders in
A. tumefaciens could therefore likely be generalized to other
species delineated with the same threshold. Similarly, the
log-linear relationship between the recombination frequency and sequence divergence found with A. tumefaciens
has also been found in several other models, thus indicating
that the results obtained with this taxon could even be generalized to other taxa displaying a similar relationship. In
addition, A. tumefaciens allowed us to finely study patterns
between very closely related genomic species. The present
study performed with a representative marker gene thus
suggested that there is very little difference in horizontal
gene transfers within and between very closely related
but heterogeneous species in bacteria. Sequence diversity
as a mechanism factor for the efficiency of the homologous
recombination mechanism that is thought to actually
control sexuality—whereby genes are integrated in the
chromosomal genome and transmitted to the progeny—appears thus to play little role in the genetic cohesion of bacterial species.
Our results thus support the conclusions of Hanage
et al. (2006) that ‘‘empirical estimates of the relationship
between sequence divergence and recombination rate indicate that the decline in recombination is an insufficiently
steep function of genetic distance to generate species.’’ Obviously, sexual isolation of species can be experimentally—
although often virtually—performed to circumscribe
eukaryote species. The present findings indeed demonstrate
that this is not the case for bacteria. Moreover, and contrary
to what happens in eukaryotes, limited but numerous genetic exchanges occurred during the very long period during which speciation occurs in bacteria. Actually, according
to the model of temporal fragmentation speciation of Retchless and Lawrence (2007), genetic isolation of bacteria may
be established at different times for different chromosomal
regions during speciation, because bacterial recombination
involves the occasional transfer of only small DNA fragments instead of complete genomes. Consequently, bacterial speciation may have lasted millions of years following
acquisition of the first niche-specific genes, a period during
which homologous recombination likely occurred. Consequently, the BSC for eukaryotes defined by Mayr (1942),
based on the occurrence of physiological barriers to sexual
reproduction, would certainly not be applicable and
Sexual Isolation of Bacterial Species 175
adaptable to the present genomic definition for a species described by Wayne et al. (1987) for prokaryotes. Nevertheless,
as highlighted by Cohan (2001) and Majewski (2001), behavioral, physical, and ecological barriers can also create effective sexual isolations. In the related genus Sinorhizobium,
Bailly et al. (2007) showed by linkage disequilibrium studies
that even in the case of bacteria living in the same biotope,
gene flow essentially occurs among strains belonging to the
same species rather than between closely related but different
species. Disconnected ecosystems likely constitute an efficient barrier to gene flow. Therefore, as genomic species
are assumed to be ecotypes by some authors (Cohan
2001), it is likely that different ecological specializations determined by niche-specific genes prevent mating and subsequent recombinations between nascent species more surely
than the RecA-dependent mechanism.
Although the authors generally agree that cohesive
evolutionary forces are necessary to generate microbial species, the fate of the current genomic species definition based
on methodological approaches or even the identification of
‘‘species-specific’’ niches is questionable. Bacterial species
were recently proposed to be metapopulation lineages contrary to a method-based concept (Achtman and Wagner
2008). With this concept, ‘‘species have just to be evolving
from other such lineages. Microbes that form distinct
groups owing to a cohesive force are metapopulation lineages and thus form species, whereas microbes without limits imposed by a cohesive force do not.’’ However, as
stipulated by the authors, microbiologists must still agree
on the criteria by which species can be recognized, and further experimental data will be necessary.
Supplementary Material
Supplementary tables and figures are available at Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
This work was supported by the ‘‘Unmasking species
specific genes and functions involved in species individualisation in ecotypes’’ Bureau des Ressources Génétiques
program and a personal grant from INRA-DSPE for
X.N.. D.C. received a PhD grant from the French MENESR. We are grateful to Andreas Schlüter for donating
plasmid pK18mob, and we also thank Julien Gouré for
sending the E. coli S17-1 strain. We are grateful to Philippe
Oger, Patrick Mavingui, and Pascal Simonet for helpful discussions and suggestions, and Dr Amy Sapkota from Maryland University and translator Dr D. Manley for reading the
manuscript and providing suggestions. We also thank David Chapulliot for skillful technical assistance.
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Accepted October 11, 2008