Vol. 173, No. 22JOURNAL OF BACTERIOLOGY, Nov. 1991, p. 7257-7268
Copyright © 1991, American Society for Microbiology
Recombination in Escherichia coli and the Definition of
DANIEL E. DYKHUIZENl* AND LOUIS GREEN2
Department ofEcology and Evolution, State University ofNew York at Stony Brook, Stony Brook,
New York 11794-5245,1 and Department ofMolecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado 803092
Received 19 April 1991/Accepted 10 September 1991
The DNA sequence of part of the gnd (6-phosphogluconate dehydrogenase) gene was determined for eight
wild strains of Escherichia coli and for Salmonella typhimurium. Since a region of the trp (tryptophan) operon
and thephoA (alkaline phosphatase) gene have been sequenced from the same strains, the gene trees for these
three regions were determined and compared. Gene trees are different from species or strain trees in that a gene
tree is derived from a particular segment ofDNA, whereas a species or strain tree should be derived from many
such segments and is the tree that best represents the phylogenetic relationship ofthe species or strains. Ifthere
were no recombination in E. coli, the gene trees for different genes would not be statistically different from the
strain tree or from each other. But, if the gene trees are significantly different, there must have been
recombination. Methods are proposed that show these gene trees to be statistically different. Since the gene
trees are different, we conclude that recombination is important in natural populations of E. coli. Finally, we
suggest that gene trees can be used to create an operational means of defining bacterial species by using the
biological species definition.
In bacteria, reproduction and exchange of chromosomal
genes are discrete and independent functions, not tied to-
gether in one process as in most animals and plants. Thus, all
reproduction in bacteria is asexual, and all genetic exchange
is due to processes other than reciprocal recombination. In
this paper, we shall continue to refer to transfer of genetic
material from one strain to another as recombination, even
though horizontal gene transfer would be more accurate,
since recombination is the term used in population genetics
to indicate the process that breaks down linkage disequilib-
rium. The amount of genetic transfer or sex will vary from
species to species, and the size ofthe exchanged fragment of
DNA will depend upon which of the three known mecha-
nisms is involved (18). For example, transduction-mediated
recombination is expected to transfer sections in the range of
tens of kilobases of DNA or a minute or two of the genetic
map, whereas conjugation can transfer regions of hundreds
to thousands of kilobases. Transformation is unlikely to be
important in Escherichia coli.
The extent to which recombination affects the genetic
structure of bacteria remains a question. If there is no
chromosomal recombination in a bacterial species, then all
individuals of a species are related by clonal descent, even
though they are phenotypically different because ofaccumu-
lated mutations. If a species is clonal, the phylogenies of
different genes from the same strains will be the same; i.e.,
genes of one strain would share the same most recent
common ancestor with genes of another strain. If, on the
other hand, recombination is important, the phylogenies of
different genes from the same strains will be different; i.e.,
the common ancestors of different genes from the same pair
of strains will be different.
Figure 1 illustrates this idea. Imagine a cell dividing into
t This paper is dedicated to the memory ofRalphV. Evans.
t Contribution no. 801 from the Graduate Studies inEcologyand
Evolution, State University of New York at Stony Brook.
two cells, each of which is the ancestor to a lineage. One
lineage gives rise to both strains A and B, and the other gives
rise to the C strain. Later, there is a cell that is the last
common ancestor of strains A and B. Then, gene X is
transferred from an ancestor ofthe C strain to an ancestor of
the B strain. To determine the relationship of the three
strains, A, B, and C, the same gene is sequenced from each
strain. The majority oftheir DNA, ifsequenced, would show
that A and B are more similar to each other than either is to
C. This would lead to the branching order seen in the open
bars of Fig. 1. If, however, gene X is sequenced, the data
would indicate a strain relationship such as that given by the
dark lines in Fig. 1, i.e., that strain B is more closely related
to strain C than to strain A. This difference is a consequence
of the fact that recombination mixes the phylogenetic rela-
tionships of the strains. Thus, if one can show that different
genes from the same strains have statistically different
phylogenies, either in the branching order as in the example
above or in the time of the last common ancestor as judged
by the relative rates of accumulated base pair change, then
the result indicates that recombination is an important pa-
rameter in creating the observed distribution ofgenotypes in
Over the last few years much information has accumulated
to support the clonal model of the population structure ofE.
coli (1, 30, 35). This model suggests that chromosomal
recombination is restricted in nature to such a degree that
individual cells lines persist as stable clones over long
periods of time, so long that members of the same clone can
be found around the world. Certain pathogenic clones, in
which the various isolates are indistinguishable by electro-
phoresis, biotyping, and serotyping, have a world-wide
distribution and have been isolated over the last 40 years (1,
2, 28). Evidence of the clonal population structure of E. coli
as a whole is indicated by the strong linkage disequilibrium
among many of the 12 enzyme loci analyzed in 302 electro-
phoretic types representing 1,690 isolates (29, 46).
Even ifthere is a significant but low rate ofrecombination,
DYKHUIZEN AND GREEN
FIG. 1. Effect of recombination on the apparent phylogenetic
relationship of three strains. The gene tree of gene X will put strains
B and C together, whereas the gene tree for any other gene will place
strains A and B together. This difference in the gene trees is caused
by the transfer of gene X from an ancestor of strain C into an
ancestor of strain B.
E. coli can have a clonal population structure if the species
often undergoes purifying or periodic selection (17). If peri-
odic selection is common, then one would expect to find a
limited number of isolated clones when the DNA sequences
are compared. The sequence results of Milkman and Craw-
ford (22) for the trpABC region support this expectation. The
sequences can be divided into three groups based on simi-
larity. Nine E. coli stains were identical or differed at only a
single base from the K-12 sequence. Three strains were
identical to each other and different from K-12 at 10 bases.
The last strain was different from the K-12 sequence at 44
bases. This result suggests that E. coli is composed of a
relatively limited number of geographically widespread
clones and that recombination plays little if any role in the
genetic structure of E. coli. The prediction from this model
of the population structure of E. coli is that the same DNA
phylogeny should be found for any segment of the chromo-
To test this hypothesis, we cloned and sequenced 770
bases in the middle of the gnd (6-phosphogluconate dehy-
drogenase) gene for eight of the E. coli strains that were
sequenced for trp and the homologous region of gnd from
Salmonella typhimurium (9). The data from these two genes,
plus additional data on the sequence of the alkaline phos-
phatase (phoA) gene from the same strains, is analyzed by
using statistical methods to determine the likelihood of
recombination. This analysis has implications for the way
species can be defined in bacteria.
MATERIALS AND METHODS
Strains. E. coli RM39A, RM191F, RM201C, RM217T,
RM45E, RM70B, RM224H, and RM2021 were obtained from
R. Milkman (22). They are included in the ECOR collection
(27) as numbers 4, 16, 45, 67, 69, 70, 68, and 65, respectively.
We confirmed that the strains from R. Milkman and the
comparable strains from the ECOR collection are the same
strains by comparing mobility of 6-phosphogluconate dehy-
drogenase on cellulose-acetate strips (20) and by comparing
the insertion sequence pattern on Southern blots (33). The S.
typhimurium LT2 used was obtained from J. Roth.
Cloning and sequencing. The DNA sequence ofgnd for the
K-12 strain was obtained from R. E. Wolf (23). The sequence
used represents positions 405 through 1172 in the numbering
system of Nasoff et al. (23). Southern blots of restriction
enzyme digests of genomic DNA from the nine strains were
probed with the gnd fragment, which was a 2.9-kb BglII-
EcoRI fragment covering the 1.4-kb gnd gene. This probe
was isolated from pMN4, which was provided by R. E. Wolf
(24). Restriction enzymes that yielded single hybridizing
fragments between 3 and 8 kb in size were used for prepar-
ative digests ofgenomic DNA. For each allele, fragments of
about the size of the gnd fragment were isolated from
agarose gels and cloned into pBR322. Transformants were
selected for gnd enzyme function (growth on gluconate).
Figure 2 shows the restriction maps of these clones. The
allele numbers refer to the strains from which the alleles
were obtained. The gnd alleles are aligned and oriented such
that the direction of synthesis is from left to right. All of the
alleles contain a PstI site at nucleotide 1172 (numbering as in
reference 23). Thus this site was used for subcloning into
M13, and all of the clones were oriented such that this site
was adjacent to the universal primer. Two alleles contained
PstI sites at nucleotide 405, defining a sequence of770 bases
within gnd which was used for the analysis. The other alleles
were subcloned into M13 by using convenient restriction
sites so that the homologous 770 bases could be sequenced in
all alleles. The LT2 strain had no convenient sites, and two
subclones had to be used.
Sanger dideoxy sequencing was accomplished initially
with an M13 universal primer and later with custom-made
oligonucleotides. The sequence of the K-12 allele was com-
pared with the new sequence generated, checking each
difference. About 20% was sequenced from both directions.
No errors were found when the two strands were compared.
Tree construction and testing. Gene phylogenies were
constructed as follows. The percent divergence (p) of DNA
sequence for each pair of strains was calculated and con-
verted to the Jukes-Cantor distance (d), which corrects for
multiple changes at the same site. The formula for this
distance measure and its variants were published by Nei
(25). The distance gene trees were constructed by the
unweighted pair-group method with the arithmetic mean
(25). The standard errors of the branch nodes were calcu-
lated by the method of Nei et al. (26).
The parsimony gene trees were constructed by using the
phylogenetic analysis program PAUP (version 2.3.2; David
Swofford, Illinois Natural History Survey, Champaign) and
rooted by using the Salmonella sequence as the outgroup.
The statistical comparison of different gene trees was done
by using the method of Templeton (45) as modified by
Felsenstein (11). This test, in its simplest form, is a binomial
sign test of whether there are more characters supporting
one tree over another, with the null hypothesis being that
any character is equally likely to support either of the two
A statistical test for the difference in time to the last
common ancestor is based on the assumption that branch
length is a measure of time and that the rates of change are
the same for different genes. In this test which is based on
the t test, the percentage change (p) is used. The distance (d)
could also have been used. Since the variance of p is a
function of p, the variable has to be transformed such that
the variance is constant and independent. Therefore the
statistic used is
RECOMBINATION IN E. COLI AND DEFINITION OF SPECIES
=;;;j pBR clones
FIG. 2. Restriction map ofclones ofvarious alleles ofgnd. The large thick arrow shows the direction oftranscription. The segments cloned
into pBR322 are shown as thick bars, with the region containing gnd darkened. The regions subcloned into M13 for sequencing are indicated
by the double-headed arrows.
/820.5 (1/n1 +
wherePi,is the proportion of nucleotides different between
strain pair i for gene 1, P2is the proportion for the same pair
of strains for gene 2, and n1 and n2 are the numbers of
nucleotides sampled for genes 1 and 2, respectively (42).
Since we do not know whether even the arcsin-transformed
percentages will follow a t distribution, given that they were
derived from an evolutionary process rather than indepen-
dent sampling from a constant distribution, we will call these
There will be n(n
strains. We expect some number of comparisons to be
significant by chance. The critical value can be adjusted so
that one has confidence at a certain level that comparisons
are significant. With multiple tests, the equation ofSidak (38)
is used to adjust the significance values:
- 1)/2 of these pseudo-t tests for n
a' = 1 -(1
where k is the number of comparisons and a is the desired
significance level (usually 0.05). This measure assumes that
the tests are independent, which in this case they are not.
Nucleotide sequence accession numbers. The nucleotide
sequence data in this paper have been submitted to Gen-
Bank. The primary strain numbers are the ECOR numbers
with the RM numbers as isolate number. The accession
numbers are as follows: M64324 for ECOR4 (RM39A),
M64325 for ECOR16 (RM191F), M64326 for ECOR45
(RM201C), M64327 for ECOR67 (RM217T), M64328 for
ECOR69 (RM45E), M64329 for ECOR70 (RM70B), M64330
for ECOR68 (RM224H), M64331 for ECOR65 (RM2021),and
M64332 for S. typhimurium LT2.
Sequences. The sequences are given in Fig. 3. There are
about 103 chi sites in the genome of E. coli or one about
every 5,000 bp (39). These sites promote recombination. No
chi sites were found in or around the sequenced gndalleles.
Intragenic recombination. The procedure used to deter-
* Hind II
V Kpa I
VOL. 173, 1991
DYKHUIZEN AND GREEN
SAL. .T2 -S
3000 - A?
FIG. 3. DNA sequence for a 770-bp internal region of gnd. Only nucleotides that are different from the one found in K-12 at the site are
listed. Thus sites such as 745, where K-12 contains a T and all other strains contain a C, are sites where the mutation presumably happened
in the lineage to the K-12 strain, and the T is a derived character state. The predicted amino acid sequences are different for every strain. The
amino acid changes were listed by Sawyer et al. (34). The characteristics of the synonymous site polymorphisms were presented by Sawyer
mine the importance of recombination in E. coli is to
compare the trees created from different genes in the same
strains. This procedure is based on the assumption that the
sequences have evolved independently, i.e., that there is no
creases the resolution of these tests, since it blends se-
quences together until each is about the same distance from
all others. Thus the relative rates of intragenic versus
intergenic recombination are important. If recombination
typically introduces pieces considerably larger than the
region of each gene sequenced, then the frequency of
intragenic recombination will be less than the frequency of
recombination between two well-spaced genes.
The presence of intragenic recombination has been shown
for both gnd (4, 32) and phoA (7) sequences, where short
sequences of 200 to 500 bp have been inserted, and for the
region near trp (44), where a unique deletion and a unique
rearrangement are found in all possible combinations. In
addition, although the variation at the trp locus is too low to
analyze the data statistically, the variation seems to be
clustered as if there are either hot spots of mutation (22) or
intragenic recombination. It is clear that intragenic recom-
bination occurs, but is it an amount sufficient to invalidate
the use of tree-building algorithms?
To answer this question, pairwise G tests for goodness of
fit were performed on the gnd data. The sequence was
broken into 12-bp blocks, giving 64 blocks. The numbers of
blocks for each pair of sequences with zero, one, two, three,
four, or five and greater differences were determined. The
expected number of blocks in each class was estimated by
determining the total number of differences and distributing
these into blocks by the Poisson process. The determined G
values are given in Table 1. For individual values, the
chi-square value for five degrees of freedom at 5% signifi-
cance is 11.07. Only 2 of the 45 values are above this value,
which is the expected frequency of false-positives. Since
there are multiple nonindependent tests, the critical value for
significance was adjusted by using equation 2 to 20.3 for k =
n(n - 1)/2 and 16.3 for k = n. No values are significant, and
this implies that the changes along the sequence are homo-
geneous enough to treat the sequences as independent
lineages for the purpose of estimating gene trees.
Evidence of intergenic recombination. Table 2 gives the
percent differences between pairs of strains for gnd and for
trp. The percent divergence between the Salmonella se-
quence and the E. coli sequences for trp is between 16.2 and
16.7% with an average of 16.5%; for gnd it is between 14.7
and 18.3% with an average of 15.7%. This shows that these
genes are evolving at about the same rate. If anything, trp
seems to be changing a little faster than gnd. Thus the
generally smaller differences between strains of E. coli for
RECOMBINATION IN E. COLI AND DEFINITION OF SPECIES
TABLE 1. G values testing for intragenic recombination in gnd
G value for intragenic recombination in gnd
aS. typhimurium strain.
trp than gnd cannot be explained by the rate of evolution of
gnd being faster than that of trp.
The consistency of the distances between the Salmonella
allele and the various alleles in E. coli suggest that the rates
of evolution within E. coli have been the same for all strains
(with the exception ofthe gnd of strain RM191F; see below).
This means that the branch lengths will be about equal and
that the unweighted pair-group method with the arithmetic
mean can be used to approximate the true tree. The percent
differences were converted to distances, and the trees for
gnd and trp were derived with standard errors of the node
position. It thus seems clear from comparing these trees
(Fig. 4) that multiple recombinations have taken place. Not
only is the order of branching different between the trees,
but also the distances are different. Thus different genes
within the same pair of strains must have had different cells
as the last common ancestors. They could only have been
brought into the same strains by recombination.
Statistical analysis of trees to determine intergenic recom-
bination. Although in the case above recombination seems
the only explanation for the differences in gene trees, this
will not always be the case, since the genetic divergence of
DNA depends upon chance events. The gene trees for two
genes can be quite different because ofchance convergences
and chance variations in rates. Thus, statistical analysis
must be done to determine when the differences between
gene trees are too large to explain by chance and must have
been caused by recombination. We present three ways of
analyzing gene trees to show that intergenic recombination
must be an explanation for the differences between trees.
(i) Sets of strains for each gene with statistically different
ancestors. When there is no information about similarity in
the rate of evolution for the different genes, a method that
depends upon determining which nodes are significantly
different from each other and which are not can be used. For
example, in Fig. 4 the error bars on nodes 4 through 9 for the
gnd tree overlap. All of these strains can be considered as
having the same common ancestor, and the tree can be
represented as seven lines radiating from a common point.
In this manner, strains can be divided into groups that
could have had the same common ancestor. The groups are
defined by nodes that are not significantly different from
each other. By this criterion, the gnd sequences form two
groups. One group contains the genes from strains RM39A,
LT2, and RM191F, whereas the other group contains the
genes from all other strains. The trp sequences form four
groups. The first group includes the trp sequences from
RM191F, K-12, RM201C, RM39A, and RM217T. The sec-
ond group contains the trp sequences from strains RM70B,
RM45E, and RM224H. The third group contains only the trp
sequence from strain RM202I, and the last group contains
only the trp sequence from S. typhimurium LT2. If there is
no recombination, the trp and gnd groups should match.
However, the trp groups divide and combine the gnd groups
The distance tree forphoA (Fig. 6) is derived from the data
ofDuBose et al. (7). Note that the scale is much different for
this tree. This tree is more complex, so a systematic method
of group creation has to be formalized. The phoA alleles
from strains K-12, RM191F, and RM201C form a group. This
branch is then eliminated, and consequently node 4 is
eliminated. The error bars for nodes 5 and 6 overlap, but
TABLE 2. Divergence between pairs of strainsa
% Differences between pairs
aValues in boldface type are for gnd, and those in lightface type are for trp.
bS. typhimurium strain.
VOL. 173, 1991
DYKHUIZEN AND GREEN
0.077 0.066 0.055 0.044 0.033 0.022 0.011
Genetic Distance (D)
Genetic Distance (D)
FIG. 4. Distance trees for alleles ofgndand trp from the same set of strains. All alleles are from E. coli except the one from S. typhimurium
LT2. The bars on the nodes represent the standard errors of the branch points. There is no error bar for the node of trp from RM191F and
K-12 because the sequences are identical. Likewise for the trp sequences from strains RM70B, RM45E, and RM224H. The three stains in
italics are the only group A strains.
RECOMBINATION IN E. COLI AND DEFINITION OF SPECIES
RM201 CI IRM217T
belong to same group
FIG. 5. Diagram ofgrouping of strains that must have had different ancestors. Without recombination, these groupings should be the same
for different genes in the same set of strains.
they do not overlap the error bar for node 2. Thus the alleles
from RM45E, RM217T, and RM224H form a group. Then
this branch is eliminated, which removes node 2. This leaves
only alleles from strains RM70B, RM39A, and RM202I and
nodes 1 and 3. Since the error bars from these nodes are
nonoverlapping, these three strains are divided into two
groups, with one group containing the first two alleles and
the other containing the last allele. When this is added to the
other grouping from the other genes, phoA breaks up previ-
ous groups and joins strains that previously had been sepa-
rated. Thus groups are further divided, showing multiple
recombinations. This graphic method shows that recombi-
nation is important without requiring the assumption that all
genes must evolve at the same rate. If they do, then other
tests that test for distance can also be done (see below).
(ii) Test for significant differences in branch order with
parsimony trees. The gnd sequence data provided 119 infor-
mative sites that gave three minimum-length parsimony trees
with a length of 277 and a consistency of 0.577. The trees
obtained differed in their placement of the gnd gene from
strains K-12, RM45E, RM70B, and RM224H. The tree most
unlike the distance tree was chosen and is shown in Fig. 7.
Neither this tree nor either the other two was significantly
different from the distance tree. There are a large number of
most parsimony trp trees. For each one of these, the
minimum length for the gnd data was calculated. The one
that best fit the gnd data (that gave the smallest number of
steps) was chosen. This trp tree is significantly different from
both the parsimony and the distance gnd trees (Fig. 7). Thus,
the branching orders are significantly different for these gene
trees, implying recombination.
It is of some interest that distance and the parsimony trees
-T -r ,I
Genetic Distance (D)
0.005 0.004 0.003 0.0010.000
FIG. 6. Distance tree for phoA alleles. These are the same strains as shown in Fig. 4, with the exception of LT2. Note that the scale is
VOL. 173, 1991
7264DYKHUIZEN AND GREEN
gad Parsimony Tree
K 5 7 8 4 3 6 2
god Distance Tree
FIG. 7. Branching relationships: parsimony and disi
gnd and parsimony tree of trp. The numbers 1 through,
alleles of the following E. coli strains: 1, RM39A; 2,
RM201C; 4, RM217T; 5, RM45E; 6, RM70B; 7,
RM202I. K, E. coli K-12. The distance and parsimony
are not significantly different from each other at the 5%
tree is significantly different from both gnd trees at th
place the gnd allele from strain RM191F in ve
places. The distance tree places it outside the
allele, whereas the parsimony tree places it (strg
7) within both the Salmonella outgroup (data not
the allele from RM39A (strain 1 in Fig. 7). TI
joining method (31) is more likely to give the
than is either of the other methods (15, 43). 1
placed the gnd+ from RM191F within the alle
typhimurium and RM39A, as did the parsimony t
The longer branch length ofthe gnd+ from RM19
that the rate of evolution of the gnd+ from R
faster than expected. Consequently, the equal r;
tion ofthe unweighted pair-group method with th
mean is invalidated for this strain, and the correc
place the common ancestor of the gnd+ from R
the other gnd alleles from E. coli after the specii
(iii) Test for significant ofdifferences in distancev
ancestors. The differences in branch lengths are
the gnd and trp trees (Fig. 4). This could be
differences in the rate of evolution ofthe genes oi
les from S.
tree (Fig. 8).
t tree would
es split with
FIG. 8. gnd tree generated by the neighbor joining method.
Branch lengths reflect distances.
recombination. The statistic used (equation 1) to test for a
significant difference in time to the common ancestor for
different genes in the same strains is distributed as the
Student t with infinite degrees offreedom. The variance used
is the theoretical variance that assumes that all the changes
Table 3 shows the values from the comparison between
gnd and trp. When the value is positive the gnd distance
between the two strains is longer than the trp distance and
when the value is negative, it is reversed. The comparisons
between the Salmonella strain and the various E. coli strains
are not significant, showing that the rates of evolution for
gnd and trp alleles are not significantly different. Since most
of the values for the comparison between S. typhimurium
and E. coli are negative, the trp locus may be evolving faster
than gnd. The only positive value is the comparison between
Salmonella alleles and the alleles from strain RM191F,
confirmingthepreviousobservation that thegndallele from
this strain is evolving a little faster than expected. These
effects are very small compared with the differences in
distance between genes within the E. coli strains. Conse-
quently the differences in the distances in trp and gnd cannot
be explained by differences in the rate of evolution of the
genes. Any significant differences in distance between pairs
of strains for these genes must be because of different
common ancestors, i.e., recombination. For example, the
ancestral gene ofthe K-12 trp and the RM191F trp is recent,
since these sequences are identical; but the ancestors of the
K-12 gnd and the RM191F gnd must have diverged very long
ago, since these sequences are so different.
All the values comparing E. coli strains are positive, and
most are significant iftested individually (32 out of 36). Since
there are multiple comparisons, the significance level for 5%
for the group is 3.21. Even with this value, 30 out of 36
values are significant.
An outgroup sequence was not determined to confirm that
the rate of evolution is the same in phoA as in the other two
genes, since S. typhimurium does not contain a gene that is
homologous tophoA (8). Sharp and Li (36) have shown there
is a strong negative correlation between the rate of diver-
gence of homologous E. coli and Salmonella genes and the
codon adaptation index (37). The codon adaptation index is
a measure of the degree of codon usage bias toward codons
that are favored by highly expressed genes. The codon
adaptation index forphoA is 0.35 compared with 0.38 for the
trp region and 0.55 for gnd. This is about what is expected
for a gene that codes for a protein synthesized intermittently
or in moderate amounts. Thus we will assume that the rates
of evolution of all three genes are equivalent and that
significant tests imply recombination.
Table 4 shows the values from the comparison between
gndandphoA and betweenphoA and trp. As expected, most
of the values for the comparison between gnd and phoA are
significant. The importance of recombination may be only
for gnd. Thus the test between trp and phoA is important.
Even after correction for multiple comparisons so the critical
value is 3.21, 12 values would be considered significant,
implying multiple recombinations.
3 5 7 6 8
/ \t / /
tance trees of
8 indicate the
level; the trp
e 1% level.
ain 2 in Fig.
r because of
Recombination in E. coli. The results from this study
clearly show that there is a tremendous amount of recombi-
nation and that this recombination is important in structuring
the genetics ofE. coli. Similar results were also seen when a
gene tree for gnd for other wild strains of the ECOR
RECOMBINATION IN E. COLI AND DEFINITION OF SPECIES
TABLE 3. Comparison of the distance between gnd alleles and trp allelesa
aThe significance levels for two-tailed t tests are as follows: at 0.05, 1.963; at 0.01, 2.583; at 0.005, 2.816; at 0.001, 3.304.
bS. typhimurium strain.
collection was compared with the phenogram generated
from multilocus enzyme electrophoresis (4).
Figure 5 shows that all strains except the pair K-12 and
RM201C and the pair RM45E and RM224H have to have had
a recombination event that replaced all or most of at least
one ofthe genes. Even these exceptional pairs of stains show
evidence of recombination when distances are considered.
Tables 3 and 4 show that the percent differences are signif-
icantly different for the different genes in these pairs of
strains. For the pair K-12 and RM201C the gnd alleles are
clearly more different from each other than are either of the
alleles from the other two genes, which are not significantly
different from each other. This strongly suggests that at least
one of the gnd alleles was recombined into a strain, whereas
the other genes could represent clonal frame genes. Clonal
frame refers to those genes in a particular strain that have
not been replaced by recombination since some designated
time (21). For the pair RM45E and RM224H, the percent
difference for every pair of alleles is significant. Thus there
have to have been at least two recombination events. The
large percentage of significant values in Tables 3 and 4 shows
that most pairs of strains are separated by at least two
recombination events. This is a minimum estimate.
Even those species pairs in which there is no evidence for
different ancestors for the two genes (i.e., the percentages
are not significantly different for the two genes) may have
whether the ancestral genes could have arisen at the same
time. If the ancestral genes arose at about the same time but
in different strains, recombination would be required but
would not be identified. In addition, the record of earlier
recombinations is eliminated by later ones. Thus, rates of
recombination will be hard to estimate.
One way of estimating the rate would be to compare
strains with recent common ancestors so that the clonal
frame can be identified. This allows the number of segments
inserted by recombination to be determined and rates of
recombination to be estimated relative to the mutation rate.
There is evidence ofrecombination within a clone as defined
by all the strains having the same electrotype (16). By using
extensive data around the trp locus, a tentative recombina-
tion rate has been estimated at 7 x 10-12 replacements per
bp per generation (21). In this collection of strains, there are
three strains where the pairwise percentages for phoA and
trp are not significantly different from each other or from
zero. One should be able to use these strains to estimate
rates of recombination..However, there is something very
odd about these three strains (K-12, RM191F, and RM201C),
which makes us suppose that the recombination rate will be
higher than expected from a simple application of this
technique. Analysis of protein gel electrophoretic data and
biotype data has consistently separated E. coli group A
strains from all others (22a, 28, 35). Group A contains K-12
and ECOR strains 1 through 25. The presumption is that this
is a recently arisen clone in which the clonal frame has not
been made unrecognizable by recombination. Since K-12,
RM191F, and RM201C are very similar in two of three
genes, it could be surmised that these genes are part of the
clonal frame and thereby that all three of these strains are
group A strains. RM191F is the same as ECOR16, which is
a group A strain, but RM201C is the same as ECOR45,
which is not a group A strain. Thus, either both genes have
TABLE 4. Comparison of the distance between phoA alleles and trp and gnd allelesa
aThe significance levels for two-tailed t tests are as follows: at 0.05, 1.963; at 0.01, 2.583; at 0.005, 2.816;at0.001, 3.304. Values in boldfacetypeare forphoA
versus gnd; those in lightface type are for phoA versus trp.
VOL. 173, 1991
DYKHUIZEN AND GREEN
been moved out of the K-12 type clonal frame into an
ancestor of RM201C or so many other genes have been
moved into RM201C that it is no longer recognizable as a
group A strain. Consequently, the use of these strains to
estimate the recombination rate would underestimate the
The data presented may give an impression of a higher
recombination rate than actually exists. The gnd locus is
tightly linked to the rIb locus, which codes for the 0 antigen
(14, 35). There are over 160 0-antigen types (12), which
implies selection for diversity (i.e., rare types have a selec-
tive advantage). Thus, recombinants that brought in a rare 0
antigen and the linked gnd would be selected, giving a higher
sampling of recombinants. Obviously, more extensive study
with more loci and more strains is required to distinguish
genes influenced by either diversifying selection or purifying
selection from those which are influenced only by mutation,
drift, and recombination and to determine which genes are
likely to be indicative of clonal frames so that accurate
estimates of the recombination rate can be made.
Statistical methods. In this paper we propose various
methods for the analysis of recombination. The first two are
formulated to statistically analyze trees, with a null hypoth-
esis that trees for different genes in the same strains are not
statistically different from each other. Most proposed statis-
tical tests are designed to indicate whether the derived tree is
the true tree. The purpose ofthese tests is different. The last
test, which is a pseudo-t test, attempts to determine which
sequence pairs for the same strains but different genes show
evidence of having different ancestors.
These tests rely on assumptions that may not be valid. It
is assumed that the changes between strains are selectively
neutral and equally probable at all sites. The effects of these
assumptions on the statistics of these tests are currently
being investigated. These methods and others to be devel-
oped will have to be studied to determine their level of
significance and their power. However, the pseudo-t test
does correctly provide nonsignificant values for the differ-
ences in the gnd-trp distances between S. typhimurium and
all of the E. coli strains, given the assumption that S.
typhimurium and E. coli are different species and conse-
quently do not recombine genes. This result implies that all
of the significant values for the distances between E. coli
strains are real and notjust a result ofthe departure from the
assumptions of the t-test.
An operational definition for biological species. The two
principal definitions of species are the phenetic definition
(41) and the biological species definition (19). The phenetic
or phenotypic definition distinguishes species as groups of
organisms that form a compact unit in character space, well
separated from other such groups. Traditionally, a phenetic
species is defined as a group of morphologically similar
organisms located in a particular geographical region and
morphologically distinct from other groups of organisms. In
microbiology, this definition has been extended to include
biochemical and physiological traits as well as morphological
ones. The species definition being employed by Brenner and
Falkow (5, 6), that species are groups of strains in which (i)
over 70% of their DNA reassociates under moderately
restrictive conditions and (ii) the thermal stability of this
reassociated DNA is within 4°C of that of homologous
reassociated DNA, is a phenetic definition.
Biological species are interbreeding groups of organisms,
with each species separated from others through reproduc-
tive barriers. This definition implies that the phylogenes of
different genes from individuals of the same species should
FIG. 9. Best-fit tree for 15 hypothetical strains from a group of
gene trees, which defines at least four species. See the text for a
be significantly different, whereas the phylogeny of genes
from individuals of different species should not be signifi-
cantly different. Thus we have an operational criterion for
the defining of bacterial species. Consider 15 hypothetical
strains of bacteria isolated from nature from which one is
interested in determining the number of species. Portions of
a few genes are sequenced in each strain, and gene trees are
derived. These trees are tested, and it is found that the gene
trees are inconsistent only within certain groups. For exam-
ple, the branching pattern of strains 3 through 6 (Fig. 9) is
different for different genes, but these strains are always
grouped together for all genes. Thus they would bejudged as
members of the same species and as a different species from
the other strains. Likewise strains 7 through 11 would be
judged as members of a second species, and strains 12
through 15 would be judged as members of a third species.
Since at least three strains are required to obtain inconsis-
tencies in the gene trees, this method can not determine if
stains 1 and 2 are members of the same species or represent
different species. However, a decision can be made by using
the genetic distances between strains 1 and 2 for the various
genes. One of the other strains can be chosen as the out
group, and the rates of evolution can be determined to find
out whether they are the same for the various genes. If they
are, the distances for pairs ofgenes for strains 1 and 2 can be
tested with the pseudo-t test. If the distances are signifi-
cantly different, they would be judged to be members of the
same species, whereas if the distances are not significantly
different, they would be members of different species.
In this paper we have shown that, within E. coli, the gene
trees are significantly different. It could be argued that
horizontal gene transfer between what we call different
species is so common that the method proposed in this paper
will never resolve differences between species. Therefore,
the other part of the requirement is to show that different
genes from individuals of different species provide an esti-
mation of the same tree. This was not tested directly in this
study, but there is evidence (36) that S. typhimurium and E.
coli are separate species by this definition. When homolo-
gous genes from E. coli K-12 and S. typhimurium LT2 are
compared, none are so similar that they could represent
recent horizontal transfer and the range of percent diver-
gence can be explained by different constraints on codon
usage (36). Horizontal gene transfer would destroy this
VOL. 173, 1991
RECOMBINATION IN E. COLI AND DEFINITION OF SPECIES
relationship. Therefore, these data imply that there is little
gene transfer between these species.
In this paper we are not concerned with the mechanisms of
the transition from a lattice of individual ancestors to a tree
of ancestoral species, but we recognize that during the
transition any definition of species will be difficult to apply
unambiguously. Some of the possible difficulties and com-
plications of this approach are discussed in Avise and Ball
(3). An example of a possible difficulty is seen in the various
Neisseria species. These species, which differ in sequence
by up to 23% and therefore are less similar to each other than
are E. coli and S. typhimurium, have transferred pieces of
the PBP2B gene among each other (40). The pieces trans-
ferred provide resistance to penicillin, and thus any transfer
would be strongly selected. This might represent the rare
case of horizontal gene transfer between species. Investiga-
tions with other genes may show that there is little gene
transfer, and thus these would still be considered separate
The methodologies for an operational definition of bacte-
rial species in terms of the biological species definition can
now be developed. Sequencing of DNA segments amplified
by the polymerase chain reaction (10, 13) will permit DNA
sequences for a number of genes from a large number of
strains to be acquired quickly. These can then be used to
define species by the methodology outlined above after the
proper statistical procedures have been worked out. Genes
chosen for this purpose should be chromosomal and found in
most organisms. Examples would be ribosomal genes and
genes for enzymes in central metabolism, like gnd and trp.
Genes like phoA, which are not found in many enteric
species (8), should be avoided, as should genes that provide
resistance to antibiotics, since they are more likely to have
transgressed species boundaries (40). Horizontal gene trans-
fer across species should be rare enough that it will not be a
problem. However, because of this possibility gene trees for
more than two genes should be used. The genes chosen
should be similar to characters chosen for taxonomic pur-
poses in plants and animals-those that provide traits not
important to the peculiar adaptive strategies of particular
This work was supported by Public Health Service grant
GM30201 from the National Institutes of Health.
We thank R. E. Wolf, Jr., R. Milkman, and J. Roth for strains, D.
L. Hartl, R. F. DuBose, and J. Kim for discussions, and Margaret
Riley, Judith Mongold, and Roger Milkman for their careful reading
and suggestions to improve the manuscript.
1. Achtman, M., M. Heuzenroeder, B. Kusecek, H. Ochman, D.
Caugant, R. K. Selander, V. Valsanen-Rhen, T. K. Korhonen, S.
Stuart, F. Orskov, and I. Orskov. 1983. Six widespread bacterial
clones among Escherichia coli Kl isolates. Infect. Immun.
2. Achtman, M., and G. Pluschke. 1986. Clonal analysis of descent
and virulence among selected Escherichia coli. Annu. Rev.
3. Avise, J. C., and R. M. Ball, Jr. 1990. Principles of genealogical
concordance in species concepts and biological taxonomy. Oxf.
Surv. Evol. Biol. 7:45-67.
4. Bisercic, M., J. Y. Feutrier, and P. R. Reeves. 1991. Nucleotide
sequence of the gnd gene from nine natural isolates of Esche-
richia coli: evidence of intragenic recombination as contributing
factor in the evolution of the polymorphic gnd locus. J. Bacte-
5. Brenner, D. J. 1981. Introduction to the family Enterobac-
teriaceae, p. 1105-1127. In M. P. Starr, H. Stolp, H. G. Truper,
A. Balows, and H. G. Schlegel (ed.), The prokaryotes: a
handbook on habitats, isolation and identification of bacteria,
vol. 2. Springer-Verlag, Berlin.
6. Brenner, D. J., and S. Falkow. 1971. Molecular relationships
among members of the Enterobacteriaceae. Adv. Genet. 16:81-
7. DuBose, R. F., D. E. Dykhuizen, and D. L. Hartl. 1988. Genetic
exchange among natural isolates of bacteria: recombination
within the phoA gene ofEscherichia coli. Proc. Natl. Acad. Sci.
8. DuBose, R. F., and D. L. Hartl. 1990. The molecular evolution
of bacterial alkaline phosphatase: correlating variation among
enteric bacteria to experimental manipulations of the protein.
Mol. Biol. Evol. 7:547-577.
9. Dykhuizen, D. E., and L. Green. 1986. DNA sequence variation,
DNA phylogeny and recombination. Genetics 113:s71.
10. Erlich, H. A. 1989. PCR technology: principles and applications
for DNA amplification. Stockton Press, New York.
11. Felsenstein, J. 1985. Confidence limits on phylogenies with a
molecular clock. Syst. Zool. 34:152-161.
12. Hartl, D. L., and D. E. Dykhuizen. 1984. The population
genetics of Escherichia coli. Annu. Rev. Genet. 18:31-68.
13. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990.
PCR protocols: a guide to methods and applications. Academic
Press, Inc., San Diego.
14. Jiang, X. M., B. Neal, F. Santiago, S. J. Lee, L. K. Romana, and
P. R. Reeves. 1991. Structure and sequence ofthe rfb(0 antigen)
of Salmonella serovar typhimurium (strain LT2). Mol. Micro-
15. Jin, L., and M. Nei. 1990. Limitations of the evolutionary
parsimony method of phylogenetic analysis. Mol. Biol. Evol.
16. Lawrence, J. G., D. E. Dykhuizen, R. F. DuBose, and D. L.
Hartl. 1988. Phylogenetic analysis using insertion sequence
fingerprinting in Escherichia coli. Mol. Biol. Evol. 6:1-14.
17. Levin, B. R. 1981. Periodic selection, infectious gene exchange
and the genetic structure of E. coli populations. Genetics
18. Levy, S. B., and R. V. Miller. 1989. Gene transfer in the
environment. McGraw-Hill Book Co., New York.
19. Mayr, E. 1963. Animal species and evolution. Harvard Univer-
sity Press, Cambridge, Mass.
20. Milkman, R. 1973. Electrophoretic variation in E. coli from
natural sources. Science 182:1024-1026.
21. Milkman, R., and M. M. Bridges. 1990. Molecular evolution of
the Escherichia coli chromosome. III. Clonal frames. Genetics
22. Milkman, R., and I. P. Crawford. 1983. Clustered third-base
substitutions among wild strains of Escherichia coli. Science
22a.Miller, R. D., and D. E. Dykhuizen. Unpublished data.
23. Nasoff, M. S., H. V. Baker H, and R. E. Wolf, Jr. 1984. DNA
sequence of the Escherichia coli gene, gnd, for 6-phosphoglu-
conate dehydrogenase. Gene 27:253-264.
24. Nasoff, M. S., and R. E. Wolf, Jr. 1980. Molecular cloning,
correlation ofgenetic and restriction maps, and determination of
the direction of transcription of gnd of Escherichia coli. J.
25. Nei, M. 1987. Molecular evolutionary genetics. Columbia Uni-
versity Press, New York.
26. Nei, M., J. C. Stephens, and N. Saitou. 1985. Methods for
computing the standard errors of branching points in an evolu-
tionary tree and their application to molecular datafrom humans
and apes. Mol. Biol. Evol. 2:66-85.
27. Ochman, H., and R. K. Selander. 1984. Standard reference
strains of Escherichia coli from natural populations. J. Bacte-
28. Ochman, H., and R. K. Selander. 1984. Evidence for clonal
population structure in Escherichia coli. Proc. Natl. Acad. Sci.
29. Ochman, H., T. S. Vhittam, D. A. Caugant, and R. K. Selander.
1983. Enzyme polymorphism and genetic population structure
7268 Download full-text
DYKHUIZEN AND GREEN
in Escherichia coli and Shigella. J. Gen. Microbiol. 129:2715-
30. Orskov, F., and I. Orskov. 1983. Summary ofa workshop on the
clone concept in the epidemiology, taxonomy, and evolution of
the Enterobacteriaceae and other bacteria. J. Infect. Dis. 148:
31. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol. Biol.
32. Sawyer, S. A. 1989. Statistical tests for detecting gene conver-
sion. Mol. Biol. Evol. 6:526-538.
33. Sawyer, S. A., D. E. Dykhuizen, R. F. DuBose, L. Green, T.
Mutangadura-Mhlanga, D. F. Wolczyk, and D. L. Hartl. 1987.
Distribution and abundance of insertion sequences among nat-
ural isolates of Escherichia coli. Genetics 115:51-63.
34. Sawyer, S. A., D. E. Dykhuizen, and D. L. Hartl. 1987. Confi-
dence interval for the number of selectively neutral amino acid
polymorphisms. Proc. Natl. Acad. Sci. USA 84:6225-6228.
35. Selander, R. K., D. A. Caugant, and T. S. Whittam. 1987.
Genetic structure and variation in natural population of Esche-
richia coli, p. 1625-1648. In F. C. Neidhardt, J. L. Ingraham,
K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger
(ed.), Escherichia coli and Salmonella typhimurium: cellular
and molecular biology. American Society for Microbiology,
36. Sharp, P. M., and W. H. Li. 1987. The rate of synonymous
substitution in enterobacterial genes is inversely related to
codon usage bias. Mol. Biol. Evol. 4:222-230.
37. Sharp, P. M., and W. H. Li. 1987. The codon adaptation
index-a measure of directional synonymous codon usage bias,
and its potential applications. Nucleic Acids Res. 15:1281-1295.
38. Sidak, Z. 1967. Rectangular confidence regions for the means of
multivariate normal distributions. J. Am. Stat. Assoc. 62:626-
39. Smith, G. R. 1983. General recombination, p. 175-209. In R. W.
Hedrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg (ed.),
Lambda II. Cold Spring Harbor Laboratory, Cold Spring Har-
40. Smith, J. M., C. G. Dowson, and B. G. Spratt. 1991. Localized
sex in bacteria. Nature (London) 349:29-31.
41. Sneath, P. H., and R. R. Sokal. 1973. Numerical taxonomy.
W. H. Freeman & Co., San Francisco, Calif.
42. Sokal, R. R., and F. J. Rohlf. 1969. Biometry, 1st ed., p.
607-610. W. H. Freeman & Co., San Francisco, Calif.
43. Sourdis, J., and M. Nei. 1988. Relative efficiencies of the
maximum parsimony and distance-matrix methods in obtaining
the correct phylogenetic tree. Mol. Biol. Evol. 5:298-311.
44. Stoltzfus, A., J. F. Leslie, and R. Milkman. 1988. Molecular
evolution ofthe Escherichia coli chromosome. I. Analysis ofthe
structure and natural variation in a previously uncharacterized
region between trp and tonB. Genetics 120:345-358.
45. Templeton, A. R. 1983. Phylogenetic inference from restriction
endonuclease cleavage site maps with particular reference to the
evolution of humans and apes. Evolution 37:221-244.
46. Whittam, T. S., H. Ochman, and R. K. Selander. 1983. Multi-
locus genetic structure in natural populations of Escherichia
coli. Proc. Natl. Acad. Sci. USA 80:1751-1755.