BACKGROUND
brucella is a genus of bacteria causing brucellosis, a zoonosis that affects a large variety of mammals and that is readily transmitted to humans.
the genus includes several classical species that can be distinguished by their preferential host range, surface structure, biochemical and physiological features, and genetic markers.
this classification is reflected in some degree of genetic polymorphism, one of the main sources of which is the copy number and distribution of is <dig>   <cit> .
b. melitensis and b. suis contain seven complete is <dig> copies  <cit> .
b. abortus carries six complete and one truncated is <dig> copies  <cit> , b. ceti and b. pinnipedialis more than  <dig> copies  <cit>  and b. ovis  <dig> copies  <cit> .
is <dig> is very stable: its mobility has been demonstrated only by using a "transposon trap" in vitro in b. ovis and b. pinnipedialis, but not in b. melitensis and b. abortus  <cit> .
based on this stability, polymorphism at the alkb locus  <cit>  is used to differentiate b. abortus from b. melitensis, b. ovis and b. suis in the amos multiplex pcr assay  <cit> .
is <dig> stability is not only relevant for brucella typification: its mobility is implicated in the generation of genetic diversity and speciation, as shown by the distribution of is <dig> among the extant brucella species.
here we report that is <dig> transposition and the generation of the associated polymorphism takes place in b. abortus under natural conditions, when genetic drift should be limited by the selective pressure imposed by the host.
RESULTS
in a previous work with  <dig> b. abortus strains, it was found that two isolates  displayed is <dig> profiles that were different from that typical of b. abortus field strains  <cit> .
this is confirmed here by the genetic profiling summarized in table  <dig>  and by the is <dig> southern blot presented in figure  <dig>  the latter shows that, while the reference strain b. abortus  <dig> presented seven is711-carrying fragments, isolates b <dig> , and b <dig>  b <dig> and b <dig>  displayed an additional one.
it is known that rb <dig>  a lipopolysaccharide rough strain obtained from b. abortus  <dig> by multiple in vitro passages on antibiotic containing media, harbors eight copies plus an additional one that transposed into the lipopolysaccharide wboa gene  <cit> .
similarly, b. abortus  <dig>  a strain isolated more than sixty years ago and extensively replicated in different laboratories carries eight is <dig> copies  <cit> .
however, the molecular weight of x-b <dig> and x-b <dig> fragments  was different from those bearing the extra is <dig> copies in  <dig>  and rb <dig>  .
interestingly, whereas strain b <dig>  which was isolated from the same sample as b <dig>  displayed the genetic profile typical of b. abortus, strains b <dig>  b <dig> and b <dig> showed an identical profile, even though they were from successive outbreaks in the same flock .
these results show that it is possible to find b. abortus field isolates with different is <dig> distributions.
a is profiles are shown in figure  <dig>
b a, b. abortus typical pattern; b, b. abortus  <dig> pattern; c, b. ovis typical pattern.
c b <dig> and b <dig> were isolated from the same sample.
d b <dig>  b <dig> and b <dig> are strains isolated from different outbreaks in the same flock.
e np: not performed
we characterized the insertion sites in b <dig> and b <dig>  to ascertain whether they were new or already present in other brucellae.
to this end, we carried out is-anchored pcr using is711-bound primers plus a decamer of %gc similar to that of the brucella genome .
the resulting amplicons ranged from  <dig> - <dig>  kb  with a similar distribution among strains, but with an additional pcr fragment for each b <dig> and b <dig> strains.
considering that those fragments may contain part of the additional is copies plus their surrounding sequences, we cloned and sequenced the  <dig>  kb and  <dig>  kb dna amplicons of b <dig> and b <dig>  respectively, and designed flanking primers  to confirm the position of the new is copy.
as predicted for the insertion of complete is <dig> copies of  <dig> bp in length, specific pcr products of  <dig> bp  and  <dig> bp  were amplified .
we believe that an is replicative transposition is the most plausible explanation for these results.
in fact, the sequence analysis suggested that transposition had occurred by a canonical ta duplication at ytar site .
in strain b <dig>  this site was in an intergenic region between a lactate permease gene  and bruab1_ <dig>   corresponding to a  <dig> bp bru-rs <dig> element, a palindromic repeat sequence that represents a putative insertion site for is <dig>  <cit> .
in contrast, the is <dig> extra copy in b <dig>  b <dig> and b <dig> was interrupting an orf encoding a transcriptional regulator of the marr family .
similarity searches showed that the b <dig> and b <dig> sites did not match with any of the is <dig> loci previously reported for b. abortus or even with the novel is <dig> sites recently described for brucella marine mammal strains  <cit> , although the b <dig> site was found in b. ovis.
to confirm these findings and to investigate whether these sites were also present in the genomes  of the brucella species carrying a high-copy number of is <dig>  we carried out pcr assays with b. ovis, b. ceti and b. pinnipedialis dnas.
for the b12-specific is <dig>  pcr amplifications with flanking primers yielded an is-empty locus fragment .
in contrast, the pcr amplifying the b <dig> fragment yielded the predicted  <dig> bp fragment in b. ovis but not in b. ceti or b. pinnipedialis .
the x-b <dig> and x-b <dig> is <dig> sequences were nearly identical to that of is711_1a and depicted only changes in a few nucleotides .
on the basis of the high is <dig> sequence similarity across sequenced b. abortus strains, we performed a cluster analysis between the is <dig> copies of b. abortus 9- <dig> and those additional ones found in  <dig>  rb <dig>  b <dig> and b <dig> strains to get insight about their origin .
although as expected, the analysis disclosed only low sequence dissimilarity, it suggested that the new copies might derive from is711_1a.
since a previous work has shown that the is711_xa in the b. abortus alkb locus and the is711_x- <dig> in strain  <dig> are identical to is711_1a  <cit> , the inclusion of is711_x-b <dig> and is711_x-b <dig> in the same cluster supports the hypothesis that is711_1a is more active than other copies in the b. abortus genome and can transpose into new sites or even into sites shared with related species.
is transposition can disrupt genes and produce negative polar effects, but also cause beneficial changes by remodeling genomes through long range recombination  <cit> .
in the case of strain b <dig>  it is uncertain whether the intergenic position of is <dig> disturbs the expression of nearby genes.
most is <dig> studied in detail  are also located within intergenic regions showing that transposition is mostly viable when occurring into neutral sites.
however, the extra is <dig> copy in b <dig>  b <dig> and b <dig> interrupts a putative transcriptional regulator that is expressed during the late-logarithmic phase of growth in b. melitensis   <cit>  and, interestingly, these strains did not show urease activity, a factor that has been proposed to favor brucella gastrointestinal infections in mice  <cit> .
we investigated whether the marr mutation was involved in the urease-negative phenotype by constructing a b. abortus  <dig> Δmarr mutant.
this mutant displayed urease activity , suggesting that the absence of urease in b <dig>  b <dig> and b <dig> is probably caused by mutation in ure genes  <cit> .
the fact that these urease negative marr mutant strains were repeatedly isolated from aborted fetuses for at least four years questions the relevance of this factor in placental colonization and abortion induction.
research is in progress to characterize the genetic background of this urease negative phenotype.
CONCLUSIONS
in this report, we have provided evidence that is <dig> polymorphism occurs in b. abortus field strains.
the fact that such polymorphism can take place in sites shared with related species points out the relevance of a multiple-marker approach in molecular typing of brucella species.
in addition, our results suggest that the extra is copies might originate from what seems to be the most active is <dig> copy.
although the environmental signals involved in the activation of the transposase remain unknown, host-pathogen interactions may play a role.
further work is needed to elucidate if changes promoted by is transposition are associated with virulence fluctuations in this pathogen.

 brucella is a genus of bacteria causing brucellosis, a zoonosis that affects a large variety of mammals and that is readily transmitted to humans. the genus includes several classical species that can be distinguished by their preferential host range, surface structure, biochemical and physiological features, and genetic markers. this classification is reflected in some degree of genetic polymorphism, one of the main sources of which is the copy number and distribution of is. b. melitensis and b. suis contain seven complete is copies. b. abortus carries six complete and one truncated is copies, b. ceti and b. pinnipedialis more than copies and b. ovis copies. is is very stable: its mobility has been demonstrated only by using a "transposon trap" in vitro in b. ovis and b. pinnipedialis, but not in b. melitensis and b. abortus. based on this stability, polymorphism at the alkb locus is used to differentiate b. abortus from b. melitensis, b. ovis and b. suis in the amos multiplex pcr assay. is stability is not only relevant for brucella typification: its mobility is implicated in the generation of genetic diversity and speciation, as shown by the distribution of is among the extant brucella species. here we report that is transposition and the generation of the associated polymorphism takes place in b. abortus under natural conditions, when genetic drift should be limited by the selective pressure imposed by the host.  in a previous work with b. abortus strains, it was found that two isolates displayed is profiles that were different from that typical of b. abortus field strains. this is confirmed here by the genetic profiling summarized in table and by the is southern blot presented in figure the latter shows that, while the reference strain b. abortus presented seven is711-carrying fragments, isolates b, and b b and b displayed an additional one. it is known that rb a lipopolysaccharide rough strain obtained from b. abortus by multiple in vitro passages on antibiotic containing media, harbors eight copies plus an additional one that transposed into the lipopolysaccharide wboa gene. similarly, b. abortus a strain isolated more than sixty years ago and extensively replicated in different laboratories carries eight is copies. however, the molecular weight of x-b and x-b fragments was different from those bearing the extra is copies in and rb. interestingly, whereas strain b which was isolated from the same sample as b displayed the genetic profile typical of b. abortus, strains b b and b showed an identical profile, even though they were from successive outbreaks in the same flock. these results show that it is possible to find b. abortus field isolates with different is distributions. a is profiles are shown in figure b a, b. abortus typical pattern; b, b. abortus pattern; c, b. ovis typical pattern. c b and b were isolated from the same sample. d b b and b are strains isolated from different outbreaks in the same flock. e np: not performed we characterized the insertion sites in b and b to ascertain whether they were new or already present in other brucellae. to this end, we carried out is-anchored pcr using is711-bound primers plus a decamer of %gc similar to that of the brucella genome. the resulting amplicons ranged from - kb with a similar distribution among strains, but with an additional pcr fragment for each b and b strains. considering that those fragments may contain part of the additional is copies plus their surrounding sequences, we cloned and sequenced the kb and kb dna amplicons of b and b respectively, and designed flanking primers to confirm the position of the new is copy. as predicted for the insertion of complete is copies of bp in length, specific pcr products of bp and bp were amplified. we believe that an is replicative transposition is the most plausible explanation for these results. in fact, the sequence analysis suggested that transposition had occurred by a canonical ta duplication at ytar site. in strain b this site was in an intergenic region between a lactate permease gene and bruab1_ corresponding to a bp bru-rs element, a palindromic repeat sequence that represents a putative insertion site for is. in contrast, the is extra copy in b b and b was interrupting an orf encoding a transcriptional regulator of the marr family. similarity searches showed that the b and b sites did not match with any of the is loci previously reported for b. abortus or even with the novel is sites recently described for brucella marine mammal strains, although the b site was found in b. ovis. to confirm these findings and to investigate whether these sites were also present in the genomes of the brucella species carrying a high-copy number of is we carried out pcr assays with b. ovis, b. ceti and b. pinnipedialis dnas. for the b12-specific is pcr amplifications with flanking primers yielded an is-empty locus fragment. in contrast, the pcr amplifying the b fragment yielded the predicted bp fragment in b. ovis but not in b. ceti or b. pinnipedialis. the x-b and x-b is sequences were nearly identical to that of is711_1a and depicted only changes in a few nucleotides. on the basis of the high is sequence similarity across sequenced b. abortus strains, we performed a cluster analysis between the is copies of b. abortus 9- and those additional ones found in rb b and b strains to get insight about their origin. although as expected, the analysis disclosed only low sequence dissimilarity, it suggested that the new copies might derive from is711_1a. since a previous work has shown that the is711_xa in the b. abortus alkb locus and the is711_x- in strain are identical to is711_1a, the inclusion of is711_x-b and is711_x-b in the same cluster supports the hypothesis that is711_1a is more active than other copies in the b. abortus genome and can transpose into new sites or even into sites shared with related species. is transposition can disrupt genes and produce negative polar effects, but also cause beneficial changes by remodeling genomes through long range recombination. in the case of strain b it is uncertain whether the intergenic position of is disturbs the expression of nearby genes. most is studied in detail are also located within intergenic regions showing that transposition is mostly viable when occurring into neutral sites. however, the extra is copy in b b and b interrupts a putative transcriptional regulator that is expressed during the late-logarithmic phase of growth in b. melitensis and, interestingly, these strains did not show urease activity, a factor that has been proposed to favor brucella gastrointestinal infections in mice. we investigated whether the marr mutation was involved in the urease-negative phenotype by constructing a b. abortus Δmarr mutant. this mutant displayed urease activity, suggesting that the absence of urease in b b and b is probably caused by mutation in ure genes. the fact that these urease negative marr mutant strains were repeatedly isolated from aborted fetuses for at least four years questions the relevance of this factor in placental colonization and abortion induction. research is in progress to characterize the genetic background of this urease negative phenotype.  in this report, we have provided evidence that is polymorphism occurs in b. abortus field strains. the fact that such polymorphism can take place in sites shared with related species points out the relevance of a multiple-marker approach in molecular typing of brucella species. in addition, our results suggest that the extra is copies might originate from what seems to be the most active is copy. although the environmental signals involved in the activation of the transposase remain unknown, host-pathogen interactions may play a role. further work is needed to elucidate if changes promoted by is transposition are associated with virulence fluctuations in this pathogen. 
we found several b. abortus strains isolated from milk and aborted fetuses that carried additional is copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marr transcriptional regulator gene. field isolates of b. abortus, the most common species infecting cattle, typically carry seven is copies. therefore, it represents an active mechanism for the emergence of genetic diversity in b. abortus thus contributing to intra-species genetic polymorphism. our results show that the replicative transposition of is can occur under field conditions. thus far, is transposition has only been shown in vitro and only for b. ovis and b. pinnipedialis, two species carrying a high number of is copies, but never in other brucella species, neither in vitro nor in field strains. interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. sequence analyses revealed that the new copies probably resulted from the transposition of a single is copy common to all brucella species sequenced so far.
 brucellosis is a zoonosis caused by brucella spp., a group of highly homogeneous bacteria. the insertion sequence is is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. this species-associated polymorphism is used in molecular typing and identification. field isolates of b. abortus, the most common species infecting cattle, typically carry seven is copies. thus far, is transposition has only been shown in vitro and only for b. ovis and b. pinnipedialis, two species carrying a high number of is copies, but never in other brucella species, neither in vitro nor in field strains.  we found several b. abortus strains isolated from milk and aborted fetuses that carried additional is copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marr transcriptional regulator gene. interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. sequence analyses revealed that the new copies probably resulted from the transposition of a single is copy common to all brucella species sequenced so far.  our results show that the replicative transposition of is can occur under field conditions. therefore, it represents an active mechanism for the emergence of genetic diversity in b. abortus thus contributing to intra-species genetic polymorphism. 