Departament de Microbiologia i Parasitologia
Sanitàries, Divisió de Ciències de la Salut,
Universitat de Barcelona, 08028 Barcelona,1 and
Departament de Biologia Ambiental, Universitat de les Illes
Balears and Institut Mediterrani d'Estudis Avançats
(CSIC-UIB), 07071 Palma de Mallorca,2 Spain
 |
INTRODUCTION |
Pseudomonas stutzeri was
first isolated by Burri and Stutzer (6) as Bacillus
denitrificans II and named P. stutzeri by Van Niel and
Allen (47). It has an unusual colony shape and consistency
when directly isolated, being described as wrinkly, dry, and
tenaciously coherent. P. stutzeri, a gram-negative
rod-shaped bacterium that is mobile by means of a single polar
flagellum, is a nonpigmented denitrifier that liberates nitrogen gas
from nitrate, is amylase positive and gelatinase negative, and is able to grow on maltose and starch (4, 43). P. stutzeri has a wide environmental distribution but is found mainly
in soil and water. Many strains have been isolated from clinical
specimens (20). The members of the species share
physiological characteristics that make P. stutzeri of
special interest in ecological studies. This species shows high
metabolic versatility (35) including the degradation of
environmental pollutants (1, 37) and high-molecular-weight polyethylene glycols (30). P. stutzeri serves
as a model for the study of the biochemistry and genetics of
denitrification and natural transformation processes.
Pseudomonas species are grouped on the basis of rRNA-DNA
hybridization studies (31). P. stutzeri is a
nonfluorescent denitrifying species of the genus Pseudomonas
included in the rRNA group I. P. stutzeri forms a
homogeneous group within the genus Pseudomonas, with
phenotypic traits that permit description to the species level.
However, P. stutzeri is a heterogeneous species with respect to many phenotypic characteristics and DNA composition. Several studies
have demonstrated that P. stutzeri consists of a complex collection of strains that might be distributed in more than one species (2, 25, 31, 35). DNA-DNA hybridization studies (35, 43) have shown the existence of at least eight
genomic groups, called genomovars. Confirmation of this system of
internal subdivisions was reported following other approaches to
bacterial phylogeny which included 16S rRNA gene sequencing
(2), chemotaxonomic total fatty acid analysis, total
protein pattern analysis (36), and macrorestriction
fragment analysis of genomic DNA (16). The difference in
16S rRNA sequence of one of these genomic groups, together with
differential phenotypic traits, was sufficient for genomovar 6 to be
renamed the new species Pseudomonas balearica (2). Genetic relationships among most of the P. stutzeri strains used in the present study, based on molecular
typing methods (repetitive extragenic palindromic PCR, enterobacteria
repetitive inverted consensus PCR, and internally transcribed spacer
[ITS] fingerprinting), have been published previously (4,
19).
Recently 16 P. stutzeri strains (including 9 of the 42 reported here) have been analyzed by PCR-based genomic
fingerprinting procedures and multilocus enzyme electrophoresis
(MLEE). A distinct genotype of each strain, indicating great
genotypic diversity, was found within P. stutzeri
(43). However, calculation of genetic diversity and
linkage disequilibrium from MLEE results were not provided by the
authors, and the nature of genetic population structure within P. stutzeri remains unclear.
In the present study, MLEE was used to examine genetic relationships
among 43 strains identified phenotypically as P. stutzeri. One of the strains phenotypically classified as
P. stutzeri (SD29577), however, was not identified as
P. stutzeri by previously described molecular methods
(3). Two strains of P. balearica and three reference strains of closely related Pseudomonas species,
included in rRNA group I (31), were also examined by MLEE.
All strains of P. stutzeri had been classified previously in
genomovars by DNA-DNA hybridizations or 16S-23S ITS1 restriction
fragment length polymorphism analysis (19), except CLN100,
A122/76, and A147/68, which could not be assigned to any known
genomovar. The results of our analysis indicate that the P. stutzeri population exhibits very high genetic diversity and
significant linkage disequilibrium, which implies a basic clonal
population structure.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The bacterial
strains used in this study were originally obtained from culture
collections or were isolated as indicated in Table
1. Some of these strains have already
been characterized physiologically and genomically (2, 4, 16, 19,
32, 35, 36, 38, 43). All strains were cultured by plating on
Trypticase soy agar (BBL, Cockeysville, Md.) at 30°C for 48 h.
Cell extracts.
Solid cultures were suspended in sterile 50 mM Na2HPO4-NaH2PO4
buffer (pH 7.5). Cell suspensions were disrupted by sonication in an
ice-water bath for four 30-s cycles with a Branson Sonifier model 250 (Branson Ultrasonics Co., Danbury, Conn.), using setting 3 and duty
cycle 50%. Cell debris was removed by centrifugation for 10 min at 13,500 rpm and room temperature (Sigma centrifuge model
201M). The supernatant fluid was dispensed in sterile
Eppendorf tubes and stored immediately at 
40°C until used.
Protein was measured by the method of Lowry et al. (24),
with bovine serum albumin (Sigma) as a standard.
Electrophoresis and running conditions for MLEE.
Discontinuous nondenaturing vertical polyacrylamide gel electrophoresis
was used for all enzymes. The acrylamide concentration in the gels was
10%/8% or 8%/5%, depending on the enzyme studied; 0.4 M Tris-HCl
(pH 8.8) resolving buffer and 0.125 M Tris-HCl (pH 6.8) stacking buffer
were used in all gels; 0.19 M Tris-glycine (pH 8.3) buffer was used for
the electrode compartments. Gels were used within 4 h of
preparation and run at 7°C. A constant voltage, depending on the
acrylamide concentration in the gel, was applied until the bromophenol
blue band reached the bottom of the gel. All strains were run at least
twice to confirm their genotype.
The following 20 enzymes were assayed: two glucose-6-phosphate
dehydrogenases (G6P-1 and G6P-2), two lactate dehydrogenases (LDH-1 and
LDH-2), two ethanol dehydrogenases (EDH-1 and EDH-2), leucine
aminopeptidase (LAP), esterases (EST), nucleoside phosphorilase (NSP),
leucine dehydrogenase (LED), two alanine dehydrogenases (ALD-1 and
ALD-2), threonine dehydrogenase (THD), two lysine dehydrogenases (LYS-1
and LYS-2), glutamate dehydrogenase-NADP (GD2), glutamate dehydrogenase-NAD (GD1), malic enzyme (ME), catechol 2,3 dioxygenase (C23O), and catechol 1,2 dioxygenase (C12O). The electrophoretic mobilities of the enzymes were determined by staining for specific enzyme activity as recommended by Selander et al. (41),
except for catechol dioxygenases. Dioxygenase activity was revealed by placing a filter paper soaked with 0.02 M Tris-HCl (pH 7) containing 0.03 M pyrocatechol (Sigma) over the gel for 3 h at 37°C. The stain solution was then poured off, the filter paper was discarded, and
the gel slice was kept at room temperature for an additional 3 h.
Catechol dioxygenases appeared on gels as well-defined brown bands. To
differentiate between C23O and C12O activities, the following procedure
was used. Cell extracts were maintained at 55°C for 10 min and then
cooled in ice. The resulting cell extracts were used to reveal C23O
activity only (15). The bands which appeared in gels with
complete cell extracts but not in gels with heat-treated cell extracts
were assigned to C12O activity. For each enzyme, distinct mobility
variants were designated as electromorphs and numbered in order of
decreasing anodal migration. Electromorphs were taken as products of
alleles at the analyzed enzyme loci. The absence of enzyme activity was
attributed to a null allele and designated 0.
To verify that the lack of enzymatic activity was due to the true
absence of the enzyme, rather than a laboratory artifact associated
with the poor quality of the cell extracts, we measured the protein
content of each lysate (ranging from 2.5 to 11.4 mg/ml). We prepared
concentrated cell lysates from several isolates (with apparent null
alleles) being repeatedly grown, in separate preparations, to a high
cell density, which never yielded detectable activity for the target
enzyme. In addition, among isolates with null alleles, gels stained for
other enzymatic activities revealed uniformly high activity. Distinct
combinations of alleles over the 20 loci assayed were assigned as
electrophoretic types (ETs).
Data treatment.
Genetic diversity
(hj) among ETs at an enzyme locus (i.e., the
probability that two isolates differ at the j locus) was
calculated from allele frequencies using the formula
hj = n(1 
pij2)/(n 1),
j = 1,2, ... m, where
pij is the frequency of the ith allele at the j locus, n is the number of
isolates or ETs, and m is the number of alleles assayed
(27, 29). Mean genetic diversity (H) was
calculated as the arithmetic mean of the hj values for all 20 loci. Clustering of the values obtained by isoenzyme electrophoresis was performed with a matrix of coefficients of genetic
distances by the unweighted pair-group method for arithmetic averages
by using the PHYLIP package (12). The genetic distances between pairs of ETs were calculated as the proportions of loci at
which dissimilar electromorphs occurred. The cophenetic correlation coefficient was calculated using NTSYS-pc, version 1.80 (34). Multilocus linkage disequilibrium was calculated on
the basis of the distribution of allelic mismatches between pairs of
ETs among all loci examined as the index of association
(IA) developed by Brown et al. (5)
and Maynard-Smith et al. (27). The ratio of the observed
variance in mismatches (Vo) to that expected at linkage equilibrium [VE = hj (1 hj)]
provides a measure of multilocus linkage disequilibrium that can be
expressed as IA = Vo/VE 1. For
populations at linkage equilibrium, Vo equals VE and IA has an expected
value of zero (5, 27). To test if
IA differed significantly from its expected
value of zero (i.e., the ratio
Vo/VE is significantly
differently from 1), a Monte Carlo randomization test with 10,000 resamplings was used. Samples of the same size as the original data set
were generated by randomly sampling alleles according to their
frequencies at each locus (11, 18). For each random
sample, Vo and VE were
calculated and the minimum (Vmin) and maximum
(Vmax) values of Vo for
the 10,000 samples were recorded. Significance was estimated as the probability of observing an
Vo/VE ratio at least as
extreme as that determined for the original data. The null hypothesis
of a random association of alleles (i.e., the population is at linkage equilibrium) was rejected if this probability was smaller than the
selected significance level. A GST statistics
was used to compare the mean genetic diversity of clinical and
environmental isolates (41). A Wilcoxon signed-rank test
with continuity correction where necessary was used to compare the
number of null alleles of samples with respect to the origin of
isolates (45). Unless otherwise stated, statistical
significance was taken to be indicated by P values of less
than 0.05. Computer programs written by T. S. Whittam
(41) and J. G. Lorén (14) were
used to calculate genetic diversity, Vo and
VE, and IA and to perform
the Monte Carlo randomizations.
| |
RESULTS |
ETs and genetic diversity.
Table
2 summarizes the allelic profiles of the
P. stutzeri and other Pseudomonas strains used in
this study. All multilocus genotypes were represented by a single
strain. In the collection of 48 isolates, all 20 loci were
exceptionally polymorphic, ranging from 8 (LAP) to 31 (EST) alleles,
with an average of 18.6 alleles per locus. The mean genetic diversity
in the sample was 0.885 (Tables 3 and 4).
In the P. stutzeri population studied, all 20 loci were also
highly polymorphic, ranging from 7 (LAP)
to 27 (EST and THD) alleles, with an average of 15.9 alleles per locus.
The mean genetic diversity was 0.876 (Tables 3 and 4). Tables 3 and 4
also show the results obtained for several population subsets studied.
No significant differences were detected in mean genetic diversity
among clinical (H = 0.844, 
2 = 176.08) and environmental (H = 0.890, 2 = 60.31) isolates (P > 0.9).
High frequency of occurrence of null alleles.
Only 8 (16.7%)
of the 48 strains studied (ETs 4, 5, 10, 11, 27, 28, 30, and 40)
presented activity for all 20 enzymes, and at least one metabolic
enzyme activity could not be detected in the cell lysates prepared from
40 (83.3%) of the organisms. Of these 40 isolates, 16 lacked
detectable activity for one enzyme, 9 lacked detectable activity for
two enzymes, and three enzyme activities were not found in lysates of 7 isolates. Moreover, cell lysates of ET 45 (P. stutzeri
A147/68), ET 36 (P. stutzeri A60/68), ET 33 (P. stutzeri A122/76), and ET 16 (P. stutzeri JM300) showed
metabolic activities for only 6, 10, 12, and 13 enzymes, respectively.
There was a significant relationship between the presence or absence of
detectable enzyme activity and the source of isolates. However, both
clinical and environmental isolates exhibited detectable enzyme
activities for all 20 enzymes studied.
Relationships among multilocus genotypes.
Genetic
relationships among the 42 ETs of P. stutzeri and the ETs of
the other species of Pseudomonas studied are shown in the
dendrogram in Fig. 1. The cophenetic
correlation coefficient of the total sample was R = 0.78. ETs differed at least at three loci, ETs 4 and 5 and ETs 10 and 11, which were separated by a genetic distance of 0.15. At a
genetic distance of 0.9, which reflects differences between groups at
approximately 14 or more loci, there were four cluster groups, numbered
I to IV.
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|
FIG. 1.
Dendrogram showing genetic relationships among P. stutzeri and related Pseudomonas strains.
Abbreviations: gv, genomovar; C, clinical; D, brackish water sediment;
I, industrial waste; M, marine; ND, not determined; O, aircraft
oil-contaminated soil; S, soil; W, wastewater treatment plant.
|
|
Group I contained both isolates of P. balearica (ETs 13 and
14), and these were separated from all Pseudomonas isolates
by a genetic distance of >0.93. The two P. stutzeri strains
of genomovar 4 (ETs 10 and 11) were separated from each other by a
genetic distance of 0.15. These strains were grouped in cluster II.
Five of the six ETs of genomovar 3 were also found in cluster II,
forming a homogeneous group; ET 32 (the remaining isolate of genomovar 3) was found in group IV.
Group III clustered ETs 36, 12, 16, 17, 33, and 45 of P. stutzeri (genomovars 2, 5, and 8 and strains CLN100, A122/76, and A147/68, respectively). At a genetic distance of 0.76, there were one
subcluster (A) and a single ET (P. stutzeri CLN100).
Subcluster A comprised three ETs of genomovars 2, 5, and 8 (ETs 36, 12, and 16, respectively) and two strains of P. stutzeri (ETs 33 and 45).
Group IV contained all ETs of P. stutzeri genomovars 1, 2 (except ET 36), and 7, an isolate identified as Pseudomonas
sp. (ET 28), and the ETs of reference strains of P. aeruginosa (ET 46), P. pseudoalcaligenes (ET 47), and
P. mendocina (ET 48). These three Pseudomonas
species and P. stutzeri were placed in the same DNA homology
group within RNA group I (31). The ETs of genomovar 5 (ETs
12 and 30) were found in groups III and IV.
The same cluster pattern was found for the P. stutzeri
population alone. There was no clear association in clusters among isolates of P. stutzeri when we considered source and place
of isolation. However, when two strains were grouped in the dendrogram at genetic distances below 0.55 (i.e., ETs 1-2, 4-5, 19-21, 26-27, 34-35, 36-45, and 10-11), each pair of strains belonged to the same
genomovar (except ET 45, which could not be assigned to any known
genomovar) and were isolated from the same source and geographic location. ETs 23 and 25 belonged to genomovar 1 and were both isolated in Mallorca, Spain, but ET 23 is an environmental strain and
ET 25 was obtained from a clinical sample. ETs 7 and 8 belonged to genomovar 3. Whereas ET 7 is a clinical strain isolated before 1952, ET 8 is a marine strain isolated in 1983.
Linkage disequilibrium analysis.
The complete set of isolates
and population subsets were analyzed for multilocus linkage
disequilibrium (Table 4). Figure 2 shows
the allele mismatch distribution among the P. stutzeri strains.
The IA, value which is an estimate of the
existence of assortative recombination for the 42 isolates of P. stutzeri studied, was 1.10 ± 0.22, which differs
significantly from zero (P < 0.0001). Moreover,
calculation of IA for clinical isolates resulted
in a value of 1.34 ± 0.29 (P < 0.0001), and it
was 1.78 ± 0.35 (P < 0.0001) for environmental
isolates. The IA of the genomovar 1 population
was 0.77 ± 0.32 (P < 0.005). To verify the
robustness of the linkage disequilibrium analysis, we performed
repetitive calculations of IA in subsets of the
total population analyzed in which the most closely related ET pairs
(up to a genetic distance of 0.5 [Fig. 1]) were stepwise eliminated.
Table 4 shows the IA values for these subsets
along with their significance. In this study, all values of
Vo exceeded the values expected in a corresponding population at linkage equilibrium
(VE). Values of Vo also
exceeded the 95% confidence limits of VE and
the Vmax and Vmin values
calculated by the Monte Carlo randomizations (Table 4), indicating that
the population structure is clonal (27).
| |
DISCUSSION |
Despite the exhaustive phenetic and molecular studies that had
been conducted on P. stutzeri, little was known about the
population genetics of isolates identified as P. stutzeri.
Data obtained by DNA-DNA hybridization (34, 42), 16S rRNA
gene sequencing (2), internally transcribed 16S-23S rRNA
gene spacer regions (19), total fatty acid analysis, total
protein pattern (36), and macrorestriction fragment
analysis of genomic DNA and DNA fingerprinting (16) have
shown that there are substantial levels of variability among natural
isolates of this species. For a thorough understanding of the
characteristics of a species, a knowledge of the genomic structure of
the population is essential, especially in studies of the population
dynamics or the colonization of habitats, in order to elucidate genetic
exchange in natural populations. MLEE records variation in chromosomal
genes and thereby enables the degree of gene transfer within species to
be estimated, permits relationships between bacterial isolates to be
determined, and allows phylogenetic frameworks to be constructed.
Therefore, we used the MLEE method to determine the genotypic
diversity and relationships among several strains, representing all
genomovars of P. stutzeri described to date with isolates
from very different sources.
High mean genetic diversity.
Study of the allelic variation of
housekeeping genes by MLEE has generated considerable data about the
genetic diversity of bacterial populations (41, 42, 48).
The amount of genetic variation in a bacterial species is roughly
10-fold greater than that of higher eukaryotes (28). Our
results show that the level of genetic diversity of P. stutzeri (H = 0.876) exceeds that of the other
bacteria studied (17, 23). This high degree of genetic diversity is consistent with data obtained by other experimental methods (2, 4, 32, 35, 43). Moreover, Sikorsky et al.
(43) determined the allozyme variation of 21 enzymes for 16 P. stutzeri strains. Using Sikorsky's data, we
calculated the mean genetic diversity (H = 0.811).
Although the latter used a low number of strains and starch gels for
the separation of the enzymes, a method which has been reported to be
less discriminatory than use of polyacrylamide gels (13,
22), Sikorsky et al. (43) also found that all 21 loci were highly polymorphic and that all multilocus genotypes
were represented by a single strain. These results agree with our
analysis of a significantly higher number of strains. We
completed our analysis by comparing two subgroups, clinical and
environmental isolates. The mean levels of genetic diversity of these
groups were not significantly different, which indicates that the
clinical isolates of P. stutzeri do not represent distinct
populations from the environmental isolates. This may have important
implications for the microbiology of P. stutzeri infections.
High frequency of occurrence of null alleles.
In addition to
the large mean number of alleles per locus, our study also demonstrated
an extremely high frequency of occurrence of null alleles. It is of
interest that these results are in agreement with those published by
Sikorsky et al. (43), who showed that 13 (81.25%) of the
16 P. stutzeri strains analyzed in their study lacked
detectable activity for at least one enzyme. Although only nine strains
of P. stutzeri and six enzymes were studied by both laboratories, the coincidence of observation of a high frequency of
occurrence of null alleles seems to confirm that the absence of
enzymatic activity was due to lack of the protein rather than an error
in manipulation. A high frequency of occurrence of null alleles has
been reported also for Helicobacter pylori, another species
with a high genetic diversity (17). Nevertheless, the frequency of occurrence of null alleles was far higher in P. stutzeri (43; this study) than in H. pylori (17). Certain characteristics of P. stutzeri that might explain the high occurrence of null alleles in
this species, including the great variation in the length of its genome
(10, 16, 32), the large size of its natural populations,
the nonconserved organization of its genome, and the chromosome
rearrangements that it frequent carries (16, 32, 43).
Several other explanations are possible for the high occurrence of null
alleles, such as the absence of all or part of the structural gene,
downregulation of enzyme production, and occurrence of
enzyme-inactivating mutations. Currently, we lack experimental data to
differentiate between these hypotheses. Significant differences in the
number of null alleles were detected among clinical and environmental
isolates. However, there was only moderate evidence against the null
hypothesis of no difference between the two populations (0.01 < P < 0.05), and we do not know the biological
significance of this observation.
Relationships among multilocus genotypes.
The deep
branching pattern of the dendrogram reveals the distant
relationships among the P. stutzeri isolates which differed in at least three loci (Fig. 1). The value of the cophenetic
correlation obtained (R = 0.78) falls into the
range (0.74 to 0.90) of most frequently occurring cophenetic
correlations reported by Sneath and Sokal (44). The
dendrogram derived in the present study shows no clear association with
respect to the source or geographic site of isolation. Both
clinical and environmental strains are distributed in
clusters II, III, and IV. We found strains from Mallorca in
clusters II, III, and IV, and we found strains from Great Britain and
from California in both clusters III and IV. Barcelona strains and
Denmark isolates were in clusters II and IV, respectively. In contrast,
if we consider the distribution in genomovars, we find a good
correlation among strains belonging to genomovars 1, 4, and 6. All 19 strains from genomovar 1 were found in cluster IV. The strains from
genomovar 4 (ETs 10 and 11) and those from genomovar 6 (ETs 13 and 14, which belonged to P. balearica species) were in clusters II
and I, respectively. Only two strains have been described in genomovars
5 and 7, and strain DSM50238 (genomovar 7) has been described
genotypically as an atypical isolate. Strain NF13 (genomovar 3, ET
32) is placed in cluster IV, while the rest of strains in genomovar 3 are in cluster II. However, strain NF13 was isolated from a sample
taken in the Pacific Ocean (39), and the other five
members of genomovar 3 were isolated in the Mediterranean area.
Strain NF13 was isolated from the Galápagos Rift hydrothermal
vents, located at depths of 2,500 to 2,600 m, and it grows at low
temperatures (e.g., 4°C). These characteristics might explain the
differences in enzymatic activity between strain NF13 and the other
strains classified in genomovar 3 by ITS fingerprinting
(4). Of the six strains included in cluster III, three
have not been classified in known genomovars and one is the sole
representative of a genomovar (JM300, genomovar 8). On the other hand,
a very good overall correlation can be found in the clustering of
strains obtained by the MLEE method and the DNA fingerprinting methods
previously applied. For example, strains 19SMN4 (ET 10) and ST27MN3 (ET
11) have the lowest genetic distance found for the strains studied and
have been proposed as very closely related clones in the genomic
analyses conducted to date (4, 16, 19); the MLEE
results now show this to be the case. The remote relationships
previously established between P. stutzeri and P. balearica (2, 3, 4, 16, 43) were confirmed
in our analysis. The two P. balearica strains included in this study appeared together in the dendrogram and distantly related (at a genetic distance of 0.93) to the P. stutzeri strains and the other Pseudomonas isolates analyzed.
Linkage disequilibrium analysis.
Some authors proposed the
existence of recombinational events to explain some of the diversity
found in P. stutzeri (43). Our results are
clear in this context and argue strongly against the presence of
electrophoretically detectable recombination by means of linkage
disequilibrium analysis. The IA value for the P. stutzeri population analyzed was 1.10 ± 0.22 (P < 0.0001). The IA values
were always significantly different from zero for all of the subgroups
studied (clinical and environmental isolates and strains classified as
genomovar 1 [Table 4]). Moreover, the stepwise elimination of the
seven most closely related ET pairs (Fig. 1) revealed that the
remaining ETs were also in linkage disequilibrium (Table 4). All of
these results strongly suggest that the population structure of
P. stutzeri sampled isolates is clonal and thus in
accordance with the hypothesis that in the P. stutzeri
populations analyzed, there is no significant level of assortative
recombination (27). Furthermore, the
IA value calculated from the data of Sikorsky et
al. (43) was 2.93 ± 0.35, which further supports
this hypothesis. The difference between the IA
values calculated from our results and those of Sikorsky et al.
(43) could be due to the different sample sizes of
P. stutzeri analyzed by the two laboratories.
Population structure of P. stutzeri.
P. stutzeri
is widely distributed in natural environments and shows a great
metabolic versatility (1, 30, 35, 37), characteristics
consistent with a large effective population size. Our results and data
of Sikorsky et al. (43) suggest the existence of very low
recombination rates in this bacterial species.
When there is a large population size and no assortative recombination,
bacterial clones will diverge freely by accumulating neutral mutations.
The occurrence in a particular population of adaptive mutations that
confer selective advantages in specific ecological situations leads to
the elimination of genetic diversity within the population but
will not prevent, in the presence of very low recombination rates,
genetic divergence between populations (9). Thus, the
exceptionally high genetic diversity of P. stutzeri could be
the result of niche-specific selection that occurs during colonization
and adaptation to a wide range of microenvironments (40).
The observations that this species is naturally competent (7, 8,
46), with the presence of insertion sequences and reports of
mosaic gene structures together with considerable variation in the
length of the genome (16), suggest that some different events may contribute to the overall species diversity. However, the
IA values obtained indicate that horizontal gene
transfer and recombination processes, if they exist, are not sufficient to disrupt the allele associations because there is still a strong linkage disequilibrium among the P. stutzeri isolates.
In conclusion, our results indicate that P. stutzeri
exhibits an exceptionally high diversity within a clonal population
structure. However, the existence of a strong linkage disequilibrium
can be explained in these cases considering that, like many bacterial species, P. stutzeri forms a metapopulation integrated by
multiple ecological populations. These populations occupy different
ecological niches, and recombination, although possible within
populations, is rare or absent between distinct populations. Such a
population structure can introduce linkage disequilibrium in a sample
of isolates from different populations (26, 33, 48).
Although more extensive studies are necessary to assess the population structure of these ecological populations of P. stutzeri,
the results reported here are consistent with the conclusion that this
bacterial species represents a good example of a phenetically cosmopolitan ecological species sensu Istock (21), i.e., a
species form characterized by a circumscribed phenotypic variation,
restricted local sets of genetic clones, and no or rare recombination.
The clonal sets are genetically diverse, but phenotypic resemblance is
sufficient to make phenetic classification and identification possible.
This bacterial species represents the highest genetic diversity
described to date. MLEE data confirm the results obtained by other
techniques to the effect that some clones of P. stutzeri are
distinct enough to warrant taxonomic differentiation (2).
We thank B. Holmes for kindly supplying strains. We are also
grateful to Maribel Farfán for her valuable contribution. We also
thank two anonymous reviewers for their helpful comments and suggestions.
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Abstract
Introduction
