BACKGROUND
bacteria in the genus brucella are the etiological agents of brucellosis, one of the most important zoonotic diseases, affecting human and domestic animals of high economic and social value.
the genus contains several species that are defined mainly on the basis of animal host specificity  <cit> .
a growing interest in development of tools for molecular epidemiology and control of brucellosis has resulted in a large amount of published genome sequence data.
the complete genome sequences of b. melitensis  <cit> , b. suis  <cit> , and two b. abortus strains  <cit>  are already published, and the genome sequences of b. ovis, b. canis and the b. abortus vaccine strain s <dig> are also completed but not published yet.
the genus brucella bears an insertion sequence  element, originally detected in brucella ovis as a repeated polymorphic dna sequence in  <dig>  <cit> .
subsequently, this repeat was proposed to be an is based on its genetic organisation, which is similar to that of some elements described before, and named as is <dig>  <cit>  or is <dig>  <cit> .
is elements are defined as small segments of dna with a simple genetic organisation and capable of inserting at multiple sites into target dna molecules.
the best known iss are those of the gram-negative bacteria, but these mobile elements have been widely reported in a variety of bacterial genera.
a compilation of the available data on insertion sequences can be found in the is database  <cit> .
is <dig> is an  <dig> bp element bounded by  <dig> bp imperfect inverted repeats.
this is contains two overlapping open reading frames, which are believed to translate into a single polypeptide by means of a - <dig> frameshift of the translation apparatus, a feature also present in other iss  <cit> .
is <dig> appears to duplicate the dinucleotide ta of the consensus target site, ytar  <cit> .
is <dig> is exclusively present in the genome of all described species of the genus brucella.
the sequenced genomes of closely related α-proteobacteria do not contain an is obviously related to is <dig>  suggesting that this insertion sequence is an element native to the genus brucella.
furthermore is <dig> has been shown to be associated with the repeated elements bru-rs <dig> and bru-rs <dig> that could represent insertion hot spots for this is  <cit> .
some published work already suggested that is <dig> could be an active transposon  <cit> .
the copy number of is <dig> is different in the species of the genus.
b. melitensis has  <dig> copies of this element; the same number as b. abortus and b. suis.
however, b. ovis contains about  <dig> copies  <cit> , and the recently discovered brucella from marine mammals carry more than  <dig> is <dig> copies  <cit> .
furthermore, the finding of a new is <dig> copy inactivating the wboa gene in the vaccine strain rb <dig> <cit>  also indicated that is <dig> could be an active element.
however, there is no experimental evidence of is <dig> transposition up to now.
due to its proven genomic stability, is <dig> has been used as a target to identify brucella species and biovars  <cit> .
other iss have been described in brucella in addition to is <dig>  such as the elements named isbm <dig>  isbm <dig> and isbm <dig>  <cit>  and isbm <dig>  <cit> , as well as is <dig>  which forms part of the composite transposon tn <dig>  <cit> , but the mobility of these elements has not been observed experimentally.
in this study we report evidence for is <dig> transposition from b. ovis and b. pinnipedialis by using pgbg <dig>  a plasmid for isolating mobile dna elements  <cit> .
this constitutes the first experimental report of activity of a transposon in the genus brucella.
RESULTS
isolation and characterisation of tetracycline resistant derivatives of pgbg <dig> in brucella
the transposon trap plasmid pgbg <dig> was introduced into brucella by conjugation as described in the materials and methods.
plasmid pgbg <dig> carries a tetracycline resistance gene whose transcription is blocked by the repressor of bacteriophage lambda.
insertions of resident transposons into the repressor gene result in transcription of the tcr gene of pgbg <dig> and can be selected on tetracycline containing media.
as a preliminary step for the transposition experiments, we determined the stability of pgbg <dig> and the mic of tetracycline for the different brucella species to be used, since this plasmid had not been employed before in this genus.
as result of these studies we found that brucella strains must be grown in the presence of chloramphenicol in order to maintain pgbg <dig>  and also determined that the most appropriate tetracycline concentration to avoid the appearance of spontaneous mutants in plasmid free brucella was  <dig> μg ml- <dig>  to assay for transposition, various independent colonies  of each brucella strain carrying pgbg <dig> were grown in appropriate media.
cultures  were then plated separately on agar plates with  <dig> μg ml- <dig> of tetracycline or with  <dig> μg ml- <dig> of chloramphenicol , and incubated at 37°c in 5% co <dig> atmosphere until growth was observed.
the frequencies of occurrence of tetracycline resistant colonies relative to chloramphenicol resistant cfu's ranged from 10- <dig> to 10- <dig>  depending on the strain .
to determine whether the appearance of tetracycline resistant colonies was due either to transposition into pgbg <dig> of a brucella transposon or to any other mutational event, selected colonies from each independent experiment were analysed by pcr using the primers g <dig> and g <dig> , which amplified the selection cartridge of pgbg <dig> plasmid as a  <dig>  kb fragment.
some of the tetracycline resistant isolates produced a pcr amplification fragment of around  <dig>  kb.
the size of these dna fragments represented an increase of approximately  <dig>  kb relative to the size of the target region of pgbg <dig>  which was consistent with the integration of a copy of is <dig> into the repressor controlling the tetracycline resistance gene of pgbg <dig>  from the four brucella strains in which the transposon trap pgbg <dig> was used, only b. ovis boc <dig> and b. pinnipedialis b2/ <dig> produced tetracycline resistant plasmids containing insertions .
no tetracycline resistant colonies containing insertions in the experiments that used b. abortus or b. melitensis as hosts were obtained.
some tetracycline resistant colonies were obtained with these two donors.
pcr analysis of the corresponding plasmids in these cases,  could not be explained by integration events.
these resistant mutants probably resulted from point mutations  or from deletions  in the pgbg <dig> selection cartridge .
* average frequency of all experiments.
the number of independent experiments is in parentheses
identification of is <dig> in pgbg <dig> tcr insertions
as a first step towards identification of the dna inserted into the tetracycline resistant insertional mutants, the pcr products were digested with the restriction enzyme haeiii.
six different restriction patterns were observed, four in b. ovis boc <dig>  and two in b. pinnipedialis b2/ <dig> .
to confirm whether the insertions were due to integration of is <dig> into pgbg <dig>  the haeiii digested pcr products containing insertions were transferred to a nylon membrane and hybridised with an is <dig> probe.
southern hybridisation analysis showed that all insertions in pgbg <dig> were due to the presence of is <dig> .
the different restriction patterns obtained with haeiii could indicate that the is had inserted in different positions of the target gene, but could also be the result of is <dig> sequence polymorphism in b. ovis and b. pinnipedialis, as both species are poorly characterised.
in order to clarify this point, the pcr products obtained from selected tetracycline resistant insertional mutants  were purified and sequenced with the isout1/isout <dig>  and the reptc1/reptc <dig> pairs of primers .
sequence analysis of the is <dig> inserted copies
twenty one pcr products from pgbg1::is <dig> tetracycline resistant mutants were selected to determine the complete sequence of the is <dig> copy which integrated into ci gene.
the sequence analysis showed that all the is <dig> insertions had taken place at the same point within the ci gene, near its 5' terminal end.
in all cases the insertion occurred at the sequence ttaa according to the predicted is <dig> target.
the central dinucleotide ta was duplicated at both sides of the element as was expected.
is <dig> insertions at this point occurred in the two possible orientations.
it was surprising to find that all the is <dig> insertions occurred in the same point of the ci gene.
transposons vary in the degree of target specificity from highly specific  to near random insertion transposons  <cit> .
the observance of a single insertion site among  <dig> independent transposition experiments indicated a strong degree of specificity in target selection.
the reported target for is <dig> integration was the very frequent ytar sequence.
however, a preference for insertion of is <dig> into the bru-rs palindromic repeats has also been reported, indicating the existence of some undetermined determinants of integration specificity.
our experimental system can only detect insertions into the  <dig> kb ci gene, which contains around fifteen ytar sites.
the reason for the observed transposition preference for a single site remains unexplained.
sequence analysis indicated the presence of four different copies of is <dig> disrupting the ci gene.
each copy was designated with a different nomenclature: is711-isoa, is711-isob, is711-isoc and is711-isod.
nucleotide sequences of these is <dig> copies were compared with the copy named is711a from b. ovis .
the copy designated as is711-isob was identical to is711a.
differences between other is <dig> copies were due to a few nucleotide substitutions that in some cases led to the appearance or disappearance of some of haeiii restriction sites .
these sequence differences were responsible of the remaining diversity in haeiii digestions shown in fig.
<dig>  some of these nucleotide changes were silent and the putative transposases of the different is <dig> copies were identical to that of is711a, with the exception of is711-isod which was different to other copies by  <dig> amino acids which were gly in place of arg, lys replacing glu and val instead of ala.
these results demonstrate that some of the is <dig> copies in b. ovis and b. pinnipedialis were transpositionally active even when the observed transposition frequency was very low.
we were unable to precisely determine transposition frequencies with this assay but if we consider that the tetracycline resistant colonies selected for analysis were representative of the whole population, we know that  <dig> % of the mutants obtained in b. ovis and  <dig> % of those obtained in b. pinnipedialis were due to is <dig> transposition .
these percentages allowed us to estimate the average transposition frequency around 10- <dig> in both species.
these frequencies are low when compared with other transposons.
transposition activity of is711
transposition of is <dig> was detected only in b. ovis and b. pinnipedialis.
we were unable to detect transposition either in b. abortus or in b. melitensis under the same experimental conditions.
many attempts to detect is <dig> transposition in e. coli from plasmids containing cloned copies of is <dig> were also unsuccessful, even when powerful detection systems as the sacb transposon trap  <cit>  were used .
all these data indicated that is <dig> transposition activity was very low, restricted to brucella maybe due to the requirement for specific host factors, and only detectable in species with a high number of copies of is <dig> per genome.
the presence of some is <dig> copies in these species encoding a full length transposase without the need for a frameshift event, could explain the observed transposition capability.
however, such a copy of is <dig> has not been observed in the genomic sequence of b. ovis released recently to genbank and a gene dosage effect is a more favoured hypothesis for transposition in b. ovis and b. pinnipedialis.
the requirement for many copies of the is could be due either to a very low level of expression of the transposase, requiring an increased dose effect for activity, or to the presence of point mutations leading to inactivity of some of the copies found in b. abortus and b. melitensis.
there are several reasons that could explain a low level of is <dig> transposase expression.
firstly, that the regular initiation codon for is <dig> orfa is gtg, a less efficient initiation codon than the canonic atg.
secondly, that a - <dig> frameshift could be necessary in order to have an active is <dig> transposase molecule.
this event is usually very inefficient with less than 5% of the translation products shifted to translate into the complete transposase.
furthermore, in some insertion sequences orfa possesses an inhibitory action on the transposition process  <cit> .
we examined the nucleotide sequence of the is <dig> copies in the genomes of the three sequenced brucella species and analysed them to detect the reasons for their inactivity.
in some of the copies either orfa or orfb were truncated or altered by single nucleotide insertions or deletions.
furthermore, we found that in many of these copies the start codon for orfa was ttg instead of gtg as happens in all the copies rescued after inactivation of the ci repressor in pgbg <dig>  ttg has been reported to be less efficient than gtg as a translation initiation codon  <cit> .
these data support that the lack of activity observed in b. abortus and b. melitensis as well as the low transposition frequency observed in b. ovis and b. pinnipedialis could be due to the low expression  of the is <dig> transposase protein.
distribution and analysis of is <dig> in the brucella sequenced genomes
the reported sequence of is <dig>  <cit> ,  was compared with the available brucella genomic sequences using local blast searches.
the results showed the presence of  <dig> is <dig> copies in b. suis and b melitensis.
b abortus 9_ <dig> also presented  <dig> is <dig> copies but one of them was severely truncated and reduced to a  <dig> bp terminal remnant.
b. abortus  <dig> contained the same is <dig> copies as the strain 9_ <dig> and one additional is <dig> copy located at exactly the same position as one of the pre-existing copies.
it is well known that the b. abortus vaccine strain rb <dig>  which is a derivative of  <dig>  contains an extra copy of is <dig> interrupting the wboa gene  <cit> .
the genome of b. ovis was known to contain a high copy number of is <dig> estimated by hybridisation in around  <dig> copies  <cit> .
the genome sequence of this species has been recently deposited in genbank .
genome sequence analysis revealed the presence of  <dig> complete copies of is <dig> in the b. ovis genome,  <dig> and  <dig> in each chromosome respectively.
upon analysis of the genomic location of the is <dig> copies we could determine that the position of six out of the seven copies was conserved in all the brucella species.
four of the conserved copies were found in the large chromosome and two in the small chromosome.
the common copies will be numbered from  <dig> to  <dig> plus a species or strain identifier .
when the nucleotide sequence of individual is copies was analyzed it was evident that some sequence divergence existed among them.
this indicated that is sequence had drifted along with the other chromosomal genes in brucella.
at the same time this sequence variation allowed us to interpret the sequence identity between is copies in the same organism as an indicator of recent transposition.
in b. abortus the extra copy 711_xa was found to be identical to 711_1a.
in b. abortus  <dig> there is an additional is <dig> copy found next to 711_3a.
this copy was also identical to 711_1a.
these observations strongly suggest that is711_1a could be active and also show the strong preference of is <dig> to transpose to some sites on the genome.
on the other hand the is <dig> copy found in the wboa gene of the b. abortus vaccine strain rb <dig> was found to be identical to 711_a <dig>  this finding was rather unexpected, since 711_a <dig> lacks the first five nucleotides from the left ir, which would usually result in loss of the transposition ability.
insertion of this copy of is <dig> into the wboa gene could also be explained by a different mobility mechanism other than transposition.
if we correlate the sequence analysis with the results presented in this study, we can conclude that the natural transposition of is <dig> occurs at very low frequency, probably due to a poor expression of is <dig> transposase.
this limitation can be overcome in b. ovis by the high copy number of is <dig>
CONCLUSIONS
in conclusion, our results demonstrate experimentally for the first time that is <dig> is an active insertion sequence of the brucellae.
on the other hand, the low transpositional activity of the sequence allows the use of this element as a tool for molecular identification, at least for the species containing a low number of copies of this insertion sequence.

 bacteria in the genus brucella are the etiological agents of brucellosis, one of the most important zoonotic diseases, affecting human and domestic animals of high economic and social value. the genus contains several species that are defined mainly on the basis of animal host specificity. a growing interest in development of tools for molecular epidemiology and control of brucellosis has resulted in a large amount of published genome sequence data. the complete genome sequences of b. melitensis, b. suis, and two b. abortus strains are already published, and the genome sequences of b. ovis, b. canis and the b. abortus vaccine strain s are also completed but not published yet. the genus brucella bears an insertion sequence element, originally detected in brucella ovis as a repeated polymorphic dna sequence in. subsequently, this repeat was proposed to be an is based on its genetic organisation, which is similar to that of some elements described before, and named as is or is. is elements are defined as small segments of dna with a simple genetic organisation and capable of inserting at multiple sites into target dna molecules. the best known iss are those of the gram-negative bacteria, but these mobile elements have been widely reported in a variety of bacterial genera. a compilation of the available data on insertion sequences can be found in the is database. is is an bp element bounded by bp imperfect inverted repeats. this is contains two overlapping open reading frames, which are believed to translate into a single polypeptide by means of a - frameshift of the translation apparatus, a feature also present in other iss. is appears to duplicate the dinucleotide ta of the consensus target site, ytar. is is exclusively present in the genome of all described species of the genus brucella. the sequenced genomes of closely related α-proteobacteria do not contain an is obviously related to is suggesting that this insertion sequence is an element native to the genus brucella. furthermore is has been shown to be associated with the repeated elements bru-rs and bru-rs that could represent insertion hot spots for this is. some published work already suggested that is could be an active transposon. the copy number of is is different in the species of the genus. b. melitensis has copies of this element; the same number as b. abortus and b. suis. however, b. ovis contains about copies, and the recently discovered brucella from marine mammals carry more than is copies. furthermore, the finding of a new is copy inactivating the wboa gene in the vaccine strain rb also indicated that is could be an active element. however, there is no experimental evidence of is transposition up to now. due to its proven genomic stability, is has been used as a target to identify brucella species and biovars. other iss have been described in brucella in addition to is such as the elements named isbm isbm and isbm and isbm, as well as is which forms part of the composite transposon tn, but the mobility of these elements has not been observed experimentally. in this study we report evidence for is transposition from b. ovis and b. pinnipedialis by using pgbg a plasmid for isolating mobile dna elements. this constitutes the first experimental report of activity of a transposon in the genus brucella.  isolation and characterisation of tetracycline resistant derivatives of pgbg in brucella the transposon trap plasmid pgbg was introduced into brucella by conjugation as described in the materials and methods. plasmid pgbg carries a tetracycline resistance gene whose transcription is blocked by the repressor of bacteriophage lambda. insertions of resident transposons into the repressor gene result in transcription of the tcr gene of pgbg and can be selected on tetracycline containing media. as a preliminary step for the transposition experiments, we determined the stability of pgbg and the mic of tetracycline for the different brucella species to be used, since this plasmid had not been employed before in this genus. as result of these studies we found that brucella strains must be grown in the presence of chloramphenicol in order to maintain pgbg and also determined that the most appropriate tetracycline concentration to avoid the appearance of spontaneous mutants in plasmid free brucella was μg ml- to assay for transposition, various independent colonies of each brucella strain carrying pgbg were grown in appropriate media. cultures were then plated separately on agar plates with μg ml- of tetracycline or with μg ml- of chloramphenicol, and incubated at 37°c in 5% co atmosphere until growth was observed. the frequencies of occurrence of tetracycline resistant colonies relative to chloramphenicol resistant cfu's ranged from 10- to 10- depending on the strain. to determine whether the appearance of tetracycline resistant colonies was due either to transposition into pgbg of a brucella transposon or to any other mutational event, selected colonies from each independent experiment were analysed by pcr using the primers g and g, which amplified the selection cartridge of pgbg plasmid as a kb fragment. some of the tetracycline resistant isolates produced a pcr amplification fragment of around kb. the size of these dna fragments represented an increase of approximately kb relative to the size of the target region of pgbg which was consistent with the integration of a copy of is into the repressor controlling the tetracycline resistance gene of pgbg from the four brucella strains in which the transposon trap pgbg was used, only b. ovis boc and b. pinnipedialis b2/ produced tetracycline resistant plasmids containing insertions. no tetracycline resistant colonies containing insertions in the experiments that used b. abortus or b. melitensis as hosts were obtained. some tetracycline resistant colonies were obtained with these two donors. pcr analysis of the corresponding plasmids in these cases, could not be explained by integration events. these resistant mutants probably resulted from point mutations or from deletions in the pgbg selection cartridge. * average frequency of all experiments. the number of independent experiments is in parentheses identification of is in pgbg tcr insertions as a first step towards identification of the dna inserted into the tetracycline resistant insertional mutants, the pcr products were digested with the restriction enzyme haeiii. six different restriction patterns were observed, four in b. ovis boc and two in b. pinnipedialis b2/. to confirm whether the insertions were due to integration of is into pgbg the haeiii digested pcr products containing insertions were transferred to a nylon membrane and hybridised with an is probe. southern hybridisation analysis showed that all insertions in pgbg were due to the presence of is. the different restriction patterns obtained with haeiii could indicate that the is had inserted in different positions of the target gene, but could also be the result of is sequence polymorphism in b. ovis and b. pinnipedialis, as both species are poorly characterised. in order to clarify this point, the pcr products obtained from selected tetracycline resistant insertional mutants were purified and sequenced with the isout1/isout and the reptc1/reptc pairs of primers. sequence analysis of the is inserted copies twenty one pcr products from pgbg1::is tetracycline resistant mutants were selected to determine the complete sequence of the is copy which integrated into ci gene. the sequence analysis showed that all the is insertions had taken place at the same point within the ci gene, near its 5' terminal end. in all cases the insertion occurred at the sequence ttaa according to the predicted is target. the central dinucleotide ta was duplicated at both sides of the element as was expected. is insertions at this point occurred in the two possible orientations. it was surprising to find that all the is insertions occurred in the same point of the ci gene. transposons vary in the degree of target specificity from highly specific to near random insertion transposons. the observance of a single insertion site among independent transposition experiments indicated a strong degree of specificity in target selection. the reported target for is integration was the very frequent ytar sequence. however, a preference for insertion of is into the bru-rs palindromic repeats has also been reported, indicating the existence of some undetermined determinants of integration specificity. our experimental system can only detect insertions into the kb ci gene, which contains around fifteen ytar sites. the reason for the observed transposition preference for a single site remains unexplained. sequence analysis indicated the presence of four different copies of is disrupting the ci gene. each copy was designated with a different nomenclature: is711-isoa, is711-isob, is711-isoc and is711-isod. nucleotide sequences of these is copies were compared with the copy named is711a from b. ovis. the copy designated as is711-isob was identical to is711a. differences between other is copies were due to a few nucleotide substitutions that in some cases led to the appearance or disappearance of some of haeiii restriction sites. these sequence differences were responsible of the remaining diversity in haeiii digestions shown in fig. some of these nucleotide changes were silent and the putative transposases of the different is copies were identical to that of is711a, with the exception of is711-isod which was different to other copies by amino acids which were gly in place of arg, lys replacing glu and val instead of ala. these results demonstrate that some of the is copies in b. ovis and b. pinnipedialis were transpositionally active even when the observed transposition frequency was very low. we were unable to precisely determine transposition frequencies with this assay but if we consider that the tetracycline resistant colonies selected for analysis were representative of the whole population, we know that % of the mutants obtained in b. ovis and % of those obtained in b. pinnipedialis were due to is transposition. these percentages allowed us to estimate the average transposition frequency around 10- in both species. these frequencies are low when compared with other transposons. transposition activity of is711 transposition of is was detected only in b. ovis and b. pinnipedialis. we were unable to detect transposition either in b. abortus or in b. melitensis under the same experimental conditions. many attempts to detect is transposition in e. coli from plasmids containing cloned copies of is were also unsuccessful, even when powerful detection systems as the sacb transposon trap were used. all these data indicated that is transposition activity was very low, restricted to brucella maybe due to the requirement for specific host factors, and only detectable in species with a high number of copies of is per genome. the presence of some is copies in these species encoding a full length transposase without the need for a frameshift event, could explain the observed transposition capability. however, such a copy of is has not been observed in the genomic sequence of b. ovis released recently to genbank and a gene dosage effect is a more favoured hypothesis for transposition in b. ovis and b. pinnipedialis. the requirement for many copies of the is could be due either to a very low level of expression of the transposase, requiring an increased dose effect for activity, or to the presence of point mutations leading to inactivity of some of the copies found in b. abortus and b. melitensis. there are several reasons that could explain a low level of is transposase expression. firstly, that the regular initiation codon for is orfa is gtg, a less efficient initiation codon than the canonic atg. secondly, that a - frameshift could be necessary in order to have an active is transposase molecule. this event is usually very inefficient with less than 5% of the translation products shifted to translate into the complete transposase. furthermore, in some insertion sequences orfa possesses an inhibitory action on the transposition process. we examined the nucleotide sequence of the is copies in the genomes of the three sequenced brucella species and analysed them to detect the reasons for their inactivity. in some of the copies either orfa or orfb were truncated or altered by single nucleotide insertions or deletions. furthermore, we found that in many of these copies the start codon for orfa was ttg instead of gtg as happens in all the copies rescued after inactivation of the ci repressor in pgbg ttg has been reported to be less efficient than gtg as a translation initiation codon. these data support that the lack of activity observed in b. abortus and b. melitensis as well as the low transposition frequency observed in b. ovis and b. pinnipedialis could be due to the low expression of the is transposase protein. distribution and analysis of is in the brucella sequenced genomes the reported sequence of is, was compared with the available brucella genomic sequences using local blast searches. the results showed the presence of is copies in b. suis and b melitensis. b abortus 9_ also presented is copies but one of them was severely truncated and reduced to a bp terminal remnant. b. abortus contained the same is copies as the strain 9_ and one additional is copy located at exactly the same position as one of the pre-existing copies. it is well known that the b. abortus vaccine strain rb which is a derivative of contains an extra copy of is interrupting the wboa gene. the genome of b. ovis was known to contain a high copy number of is estimated by hybridisation in around copies. the genome sequence of this species has been recently deposited in genbank. genome sequence analysis revealed the presence of complete copies of is in the b. ovis genome, and in each chromosome respectively. upon analysis of the genomic location of the is copies we could determine that the position of six out of the seven copies was conserved in all the brucella species. four of the conserved copies were found in the large chromosome and two in the small chromosome. the common copies will be numbered from to plus a species or strain identifier. when the nucleotide sequence of individual is copies was analyzed it was evident that some sequence divergence existed among them. this indicated that is sequence had drifted along with the other chromosomal genes in brucella. at the same time this sequence variation allowed us to interpret the sequence identity between is copies in the same organism as an indicator of recent transposition. in b. abortus the extra copy 711_xa was found to be identical to 711_1a. in b. abortus there is an additional is copy found next to 711_3a. this copy was also identical to 711_1a. these observations strongly suggest that is711_1a could be active and also show the strong preference of is to transpose to some sites on the genome. on the other hand the is copy found in the wboa gene of the b. abortus vaccine strain rb was found to be identical to 711_a this finding was rather unexpected, since 711_a lacks the first five nucleotides from the left ir, which would usually result in loss of the transposition ability. insertion of this copy of is into the wboa gene could also be explained by a different mobility mechanism other than transposition. if we correlate the sequence analysis with the results presented in this study, we can conclude that the natural transposition of is occurs at very low frequency, probably due to a poor expression of is transposase. this limitation can be overcome in b. ovis by the high copy number of is  in conclusion, our results demonstrate experimentally for the first time that is is an active insertion sequence of the brucellae. on the other hand, the low transpositional activity of the sequence allows the use of this element as a tool for molecular identification, at least for the species containing a low number of copies of this insertion sequence. 
the copy number of is varies in the genome of the different brucella species, ranging from in b. abortus, b. melitensis and b. suis to more than in b. ovis and in brucella strains isolated from marine mammals. at present, there is no experimental evidence of transposition of is but the occurrence of this element with a high copy number in some species, and the isolation of brucella strains with "ectopic" copies of is suggested that this is could still transpose. in this study we obtained evidence of transposition of is from the b. ovis and b. pinnipedialis chromosomes by using the "transposon trap" plasmid pgbg this plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. the strains b. melitensis m, b. abortus rb b. ovis boc and b. pinnipedialis b2/ all containing the plasmid pgbg were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants. tcr mutants due to is transposition were only detected in b. ovis and b. pinnipedialis strains. four different copies of is were found to transpose to the same target sequence in the plasmid pgbg this demonstrated that is are active in vivo, specially in brucella species with a high number of is copies as b. ovis and b. pinnipedialis.
 the brucella genome contains an insertion sequence element called is or is which is specific to the genus. the copy number of is varies in the genome of the different brucella species, ranging from in b. abortus, b. melitensis and b. suis to more than in b. ovis and in brucella strains isolated from marine mammals. at present, there is no experimental evidence of transposition of is but the occurrence of this element with a high copy number in some species, and the isolation of brucella strains with "ectopic" copies of is suggested that this is could still transpose.  in this study we obtained evidence of transposition of is from the b. ovis and b. pinnipedialis chromosomes by using the "transposon trap" plasmid pgbg this plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. the strains b. melitensis m, b. abortus rb b. ovis boc and b. pinnipedialis b2/ all containing the plasmid pgbg were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants. tcr mutants due to is transposition were only detected in b. ovis and b. pinnipedialis strains.  four different copies of is were found to transpose to the same target sequence in the plasmid pgbg this demonstrated that is are active in vivo, specially in brucella species with a high number of is copies as b. ovis and b. pinnipedialis. 