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Applied and Environmental Microbiology, November 2000, p. 5104-5105, Vol. 66, No. 11
0099-2240/00/$04.00+0
LETTERS TO THE EDITOR
Genetic Diversity within E. coli
 |
LETTER |
The results shown for the 13 strains from the Escherichia
coli reference (ECOR) collection that Souza et al. included "as a reference" in their recent allozyme analysis of diverse natural E. coli isolates (8) fail to validate the newly
derived phylogeny (Fig. 1 in reference 8), leave
mysterious the correspondence between this phylogeny and that of the
ECOR collection (1-5, 7), and raise serious concerns
regarding the validity of the investigators' allozyme data and the
conclusions derived therefrom (8).
In the dendrogram of Souza, et al. (Fig. 1 in reference
8), the eight ECOR group A control strains are
variously placed in the new "ancestral cluster" (along with the
four group B1 strains) and in clusters G and C (together with the sole
ECOR group D control strain), i.e., across the breadth of the tree and
in association with members of the two other ECOR groups studied. This
conflicts with previous analyses of the ECOR strains, which with few
exceptions have placed the group A strains close together, apart from
representatives of other ECOR groups, irrespective of cluster analysis
method (e.g., principal-component analysis, UPGMA, neighbor joining, or
parsimony) or type of data set (e.g., starch or cellulose acetate gel
allozyme analysis, comparative DNA sequencing, or PCR fingerprinting) (1-5, 7).
A direct comparison of allozyme data for the 13 ECOR controls as
provided by Souza et al. (8), and as obtained from the ECOR
database at the Thomas Whittam laboratory web site
(http://www.bio.psu.edu/People/Faculty/Whittam/Lab/), for the six loci at which these two data sets coincide is
informative (Table 1). Souza et al.
report more polymorphisms at five of the six loci than are documented
in the ECOR database. These discrepancies are most marked for the eight
group A ECOR strains, among which Souza et al. list twice as many
allelic variants (not counting nulls) over the six loci as does the
ECOR database (Table 1). According to Souza et al., but not the ECOR
data base, ECOR strains 5 and 8 exhibit distinct alleles for IDH, MDH,
MPI, and/or PGM as compared with the other six group A strains (data
not shown), evidence suggesting that different strains may have been
tested as ECOR 5 and 8 in the different analyses. Still, Souza et al. report more diversity even among the remaining six group A ECOR strains
than is found in the ECOR database (data not shown).
That these discrepancies are not simply a matter of cellulose acetate
gels (8) versus starch gels (ECOR database) is shown by the
data of Pupo et al. (5). Using cellulose acetate gel allozyme analysis, these investigators found the group A ECOR strains
to be quite homogeneous across 10 enzyme loci, including the six shown
in Table 1, and derived a neighbor joining tree quite similar to that
obtained by Herzer et al. using starch gels and 38 loci (3,
5). Thus, the data of Souza et al. (8), at least for
the group A ECOR strains, are of uncertain validity. Confidence in the
remainder of these investigators' allozyme data set is weakened by the
inclusion of an enzyme (ARK, arginine kinase) that is without precedent
in the E. coli allozyme literature and the exceptional
finding of two loci for ME (malic enzyme) (3, 6).
Consequently, the tree (Fig. 1 in reference 8), the genetic diversity calculations (Table 2 in reference
8), and all inferences drawn from these analyses are
suspect and provide no support for the revised understandings of
phylogenetic relationships within E. coli proposed by the
authors (8).
| |
REFERENCES |
| 1.
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Desjardins, P.,
B. Picard,
B. Kaltenbock,
J. Elion, and E. Denamur.
1995.
Sex in Escherichia coli does not disrupt the clonal structure of the population: evidence from random amplified polymorphic DNA and restriction-fragment-length polymorphism.
J. Mol. Evol.
41:440-448[CrossRef][Medline].
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| 2.
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Guttman, D. S., and D. E. Dykhuizen.
1994.
Clonal divergence in Escherichia coli as a result of recombination, not mutation.
Science
266:1380-1383[Abstract/Free Full Text].
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| 3.
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Herzer, P. J.,
S. Inouye,
M. Inouye, and T. S. Whittam.
1990.
Phylogenetic distribution of branched RNS-linked multicopy single-stranded DNA among natural isolates of Escherichia coli.
J. Bacteriol.
172:6175-6181[Abstract/Free Full Text].
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| 4.
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Lecointre, G.,
L. Rachdi,
P. Darlu, and E. Denamur.
1998.
Escherichia coli molecular phylogeny using the incongruence length difference test.
Mol. Biol. Evol.
15:1685-1695[Abstract].
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| 5.
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Pupo, G. M.,
D. K. R. Karaolis,
R. Lan, and P. R. Reeves.
1997.
Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies.
Infect. Immun.
65:2685-2692[Abstract].
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| 6.
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Selander, R. K.,
D. A. Caugant,
H. Ochman,
J. M. Musser,
M. N. Gilmour, and T. S. Whittam.
1986.
Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics.
Appl. Environ. Microbiol.
51:873-884[Free Full Text].
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| 7.
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Selander, R. K.,
D. A. Caugant, and T. S. Whittam.
1987.
Genetic structure and variation in natural populations of Escherichia coli, p. 1625-1648.
In
F. C. Neidhardt, K. L. Ingraham, B. Magasanik, K. B. Low, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 8.
|
Souza, V.,
M. Rocha,
A. Valera, and L. E. Eguiarte.
1999.
Genetic structure of natural populations of Escherichia coli in wild hosts on different continents.
Appl. Environ. Microbiol.
65:3373-3385[Abstract/Free Full Text].
|
| | | | |
James R. Johnson
Infectious Diseases (111F)
Minneapolis VA Medical Center
1 Veterans Drive
Minneapolis, MN 55417
|
| |
AUTHOR'S REPLY |
Although Escherichia coli is perhaps the best known organism
in terms of genetics and physiology, we still have very limited knowledge of the ecology of its natural populations. For this reason we
decided to organize a collection of E. coli isolates from
wild animals of different origins, orders, and diets and analyzed it in
terms of sugar utilization, antibiotic resistance, toxin production,
and plasmid profiles as well as a preliminary population genetics
analysis based on 11 loci by using MLEE in cellulose acetate membranes
(7).
Dr. Johnson is concerned with our MLEE analysis of the ECOR collection.
We are completely confident of our results because we used a double
blind method for the labeling of the strains, and we ran our samples as
many times as necessary to be sure that all strains considered to have
the same allele had exactly the same mobility. This rigorous reading
explains the presence of new alleles when small differences in mobility
were detected. The fact that we have a diversity for the ECOR different
from that in reference 5 is due to differences in
procedure. In their study, the ECOR was used as a standard, and these
strains were "forced" to have the same alleles as the ones reported
in the original work (4) (see Materials and Methods in
reference 5). This had the artificial effect of
obtaining all the readings of the gels relative to the described bands
(6). We did not use the ECOR as a standard but just as
additional strains. Besides, due to the needs of so many different
strains, we grew the strains in glucose-enriched minimal media instead
of LB. We also used a brand of cellulose acetate membranes and buffers
and enzymes different from those used by Pupo et al. (5).
Dr. Johnson is also worried about our usage of the enzyme arginine
kinase, EC 2.7.3.3. This is an allozyme (described in reference
2) that is repeatable and variable in most bacterial species that we have analyzed. This enzyme behaves as an "average" locus in E. coli (7), and its inclusion or
deletion does not change the described patterns.
Another concern of Dr. Johnson is that the ECOR collection does not
cluster in our dendrogram as previously described. We consider our
dendrogram only a visual expression of the statistical relationships of
a set of markers that are mainly (but not only) under strong genetic
control. It is not surprising that the relationships among the strains
may appear different when different sets of strains, different genetic
markers, and different distance methods are used, in particular
considering the small number of characters (11 loci) and the large
number of strains (202). We avoided naming our dendrogram a
"phylogenetic tree" as we were aware that recombination among and
within loci is an important evolutionary force in this species (1,
3). For instance, in reference 3, the ECOR group A is separated into many different groups in the finO,
trpC, and gnd trees.
We consider that neither our collection nor the ECOR collection
represents the complete ecology of E. coli, and thus each collection helps us in a different way to understand what is E. coli. Because recombination is an important evolutionary force in
this species, there is not a single phylogenetic tree for the strains,
and in consequence we can analyze them only by using statistical
descriptions of the allelic frequencies.
| |
REFERENCES |
| 1.
|
Dykhuizen, D. E., and L. Green.
1991.
Recombination in Escherichia coli and the definition of biological species.
J. Bacteriol.
173:7257-7268[Abstract/Free Full Text].
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| 2.
|
Hebert, P. D., and M. J. Beaton.
1993.
Methodologies for allozyme analysis using cellulose acetate electrophoresis.
Helena Laboratories, Beaumont, Tex.
|
| 3.
|
Lecointre, G.,
L. Rachdi,
P. Darlu, and E. Denamur.
1998.
Escherichia coli molecular phylogeny using the incongruence length difference test.
Mol. Biol. Evol.
15:1685-1695.
|
| 4.
|
Ochman, H., and R. K. Selander.
1984.
Standard reference strains of Escherichia coli from natural populations.
J. Bacteriol.
157:690-693[Abstract/Free Full Text].
|
| 5.
|
Pupo, G. M.,
D. K. R. Karaolis,
L. Ruiting, and P. R. Reeves.
1997.
Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies.
Infect. Immun.
65:2685-2692.
|
| 6.
|
Selander, R. K.,
D. A. Caugant,
H. Ochman,
J. M. Muser, and T. S. Whittam.
1986.
Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics.
Appl. Environ. Microbiol.
51:873-884.
|
| 7.
|
Souza, V.,
M. Rocha,
A. Valera, and L. E. Eguiarte.
1999.
Genetic structure of natural populations of Escherichia coli in wild hosts on different continents.
Appl. Environ. Microbiol.
65:3373-3385.
|
| | | | |
Valeria Souza
Martha Rocha
Aldo Valera
Luis E. Eguiarte
Departamento de Ecología Evolutiva
Instituto de Ecología
Universidad Nacional Autónoma de México
Apartado Postal 70-275
Coyoacán 04510 México D.F., México
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Applied and Environmental Microbiology, November 2000, p. 5104-5105, Vol. 66, No. 11
0099-2240/00/$04.00+0
