BACKGROUND
the members of the genus brucella are gram-negative bacteria that cause brucellosis, a zoonotic disease of great importance worldwide.
currently, several brucella species are recognized  <cit> .
b. abortus, b. melitensis, b. suis, b. neotomae, b. ovis, and b. canis have been known for a long time and are traditionally distinguished according to their preferential host, biochemical tests and cell surface characteristics  <cit> .
in addition, brucella strains isolated from cetaceans and pinnipeds during the last fifteen years have been grouped into b. ceti and b. pinnipedialis,  <cit> .
very recently, some brucella strains have been isolated from the common vole and a new species, b. microti, proposed  <cit> .
b. abortus, b. melitensis and b. suis have been classically subdivided into biovars according to h2s production, co2-dependence, dye sensitivity and distribution of the a and m epitopes   <cit> .
however, because these tests are difficult to standardize, molecular markers have been investigated  <cit> .
wild type b. melitensis, b. abortus, b. suis, b. neotomae, b. ceti, b. pinnipedialis and b. microti express a smooth -type lipopolysaccharide  formed by an o-polysaccharide connected to a core oligosaccharide which, in turn, is linked to lipid a, the section embedded into the outer membrane.
however, both b. ovis and b. canis lack the o-polysaccharide and, accordingly, their lps is termed rough  .
brucella lps is of great interest not only because of these species differences but also because it is the foremost diagnostic antigen and a major virulence factor  <cit> .
despite this, the structure and genetics of brucella lps is only partially understood.
the o-polysaccharide is a homopolymer of n-formyl-perosamine in α  or in α  plus α  linkages  <cit> , and these variations relate to the main serovars in brucella s species  linkage; m dominant ; or a = m ).
previous studies in b. melitensis  <dig> m and h <dig>  have identified two genetic regions involved in o-polysaccharide synthesis and translocation .
region wbo encodes two putative glycosyltransferases  and region wbk contains the genes putatively involved in perosamine synthesis , its formylation  and polymerization  , as well as those for bactoprenol priming  and o-ps translocation .
in addition, wbk contains genes  which may code for the enzymes that furnish mannose, the perosamine precursor.
intriguingly, wbkb and manbo-ag do not generate r phenotypes upon disruption  <cit> , and b. ovis and b. canis carry wbk genes despite the absence of the o-polysaccharide  <cit> .
much less is known on the brucella core oligosaccharide.
reportedly, it contains 2-keto, 3-deoxyoctulosonic acid, mannose, glucose, glucosamine and quinovosamine  <cit>  but the structure is unknown.
thus far, only three genes have been proved to be involved in core synthesis: pgm , manbcore  and wa**   <cit> .
obviously, genetic analysis encompassing a variety of strains could shed light on the differences behind the phenotypes of s and r species, confirm or rule out a role for known genes, and identify differences that could serve as serovar or biovar markers.
with these aims, wbke, manao-ag, manbo-ag, manco-ag, wbkf, wkdd, wboa, wbob, wa** and manbcore were analyzed for polymorphism in the classical brucella spp., b. ceti, and b. pinnipedialis.
RESULTS
lps genes in brucella spp.
and biovars
gene polymorphism in wbk
wbke
for all strains, the wbke pcr-amplified product displayed the same ecorv, hinfi, psti and pvuii rflp patterns.
although b. melitensis 63/ <dig> biovar  <dig> showed a different styi pattern, only one of eight additional b. melitensis biovar  <dig> strains tested showed this styi pattern .
manao-ag
b. neotomae had a distinct manao-ag restriction pattern consisting of an additional avaii site .
moreover, in silico analysis showed a specific profile for b. ovis consisting of a nucleotide substitution  at position  <dig> which modified the mana c-terminal sequence at amino acid  <dig> .
also, a single nucleotide deletion  was detected at position 738; this frame shift leads to a change in amino acid sequence after position  <dig>  nucleotide sequence of pcr products from several strains confirmed the deletion in manao-ag as characteristic of b. ovis .
species
b. melitensis
b. abortus
b. suis
b. ovis
b. canis
b. neotomae
b. pinnipedialis
b. ceti
a atcc, american type culture collection; bccn, brucella culture collection, nouzilly, france.
na: not amplified
manco-ag
despite the use of several endonucleases , manco-ag restriction patterns were identical in all brucella strains .
therefore, no polymorphism was observed by this method.
manbo-ag
b. melitensis  <dig> m  and b. abortus tulya  presented a similar manbo-ag restriction pattern , and b. melitensis biovars  <dig> and  <dig> showed a sau3a site absent in other strains .
all b. abortus ) strains tested showed a specific pattern characterized by the absence of the ecorv site at position  <dig> .
b. suis biovars  <dig>   <dig>   <dig> and  <dig>  b. canis and b. neotomae formed a separate group  on the basis of the sau3a restriction patterns of this gene.
b. ovis shared this pattern only partially because it lacked one more sau3a site .
b. suis biovar  <dig> strains lacked the manbo-ag sau3a site and showed an additional hinfi site in this gene .
when this gene was amplified  from b. ovis 63/ <dig>  sequenced, and aligned with the homologous genes of b. melitensis biovar  <dig>  b. abortus biovar  <dig>  and b. suis biovar  <dig>  polymorphism in both sequence and length was detected.
as compared to b. melitensis biovar  <dig> and b. abortus biovar  <dig>  two more nucleotides were found at position 1265– <dig> in b. suis biovar  <dig> and b. ovis which should lead to a modification of c-terminal sequence of the protein .
all strains isolated from marine mammals yielded restriction manbo-ag patterns very different from those of the six classical species  as well as a larger pcr product  .
sequencing of the pcr product of three strains  revealed an is <dig> element  inserted into the gene  , and this insertion was confirmed by pcr in  <dig> additional marine mammal strains .
wbkd and wbkf
the wbkd pcr product was tested with hinfi, avaii, sau3a, bgli, clai and styi, but a very low degree of dna polymorphism was observed .
for b. melitensis, b. neotomae and all marine mammal strains, all strains showed the same sau3a pattern.
an additional sau3a site was observed for all b. abortus, b. suis and b. ovis strains .
interestingly, the b. canis product showed a reduced size of around  <dig> bp and, therefore, yielded species specific restriction patterns.
this result indicated the existence of a deletion in b. canis wbkd .
the wbkf pcr product showed also a low degree of polymorphism when tested with ecorv, haeii, hinfi, alui, sau3a and styi .
one pattern, however, was specific for b. melitensis biovar  <dig> which lacked an alui site, and a distinct pattern for two b. abortus biovar  <dig> and 45/ <dig>  was also observed with alui site.
remarkably, no amplification was obtained for b. canis, suggesting that the sequence of the wbkf-b primer corresponded to a deletion extending from the adjacent wbkd gene .
in fact, when the appropriate primer was used, the wbkf pcr product showed a reduced size of about  <dig> bp.
to examine this point further, the wbkf-wbkd locus was amplified and sequenced in b. melitensis, b. ovis and b. canis.
the sequences showed a  <dig> bp deletion in b. canis extending from wbkd nucleotide  <dig>  to wbkf nucleotide  <dig>   as confirmed by the genome sequence of b. canis rm 6/ <dig>  .
moreover, as compared to their homologs in b. melitensis, b. abortus and b. suis, gene wbkf of b. ovis showed a single nucleotide deletion at position  <dig>  this frame shift mutation necessarily leads to an extensive modification of cognate protein .
gene polymorphism in wboa
a low degree of dna polymorphism was observed in wboa.
however, one pattern was specific of b. abortus since all strain testedlacked an alui site.
as described above, no amplification was observed for any b. ovis strain.
this confirms  <cit>  that absence of wboa  is a b. ovis species-specific marker.
polymorphism in core lps genes
despite using six restriction enzymes, all brucellae displayed the same rflp pattern for the manbcore amplicon.
in silico, the four genomes available showed low polymorphism.
a single nucleotide deletion at position  <dig> was detected in b. ovis, which should modify the c-terminal sequence of the protein .
similarly, a low degree of polymorphism was observed in wa**.
with the exception of b. suis biovar  <dig>  one psti pattern was specific of b. suis.
biovar  <dig> also lacked an avaii site, which could be considered as a biovar marker.
with hinf <dig>  a pattern was specific of b. ovis .
discussion
despite the high dna homology of brucellae, gene polymorphism and species- and biovar-specific markers have been consistently found.
concerning outer membrane molecules, both have been found in genes of proteins  <cit>  but not in the lps genes examined, all of the wbk region .
interestingly, these o-polysaccharide genes were found to be highly conserved not only in the classical s brucella species and biovars but also in b. ovis and b. canis, the two species that lack the o-polysaccharide  <cit> .
therefore, an implication of these observations is that the r phenotype of b. ovis and b. canis cannot be explained by the absence of any of those seven wbk genes.
more recently, the wbk region has been extended to include wbke, manao-ag, manbo-ag, manco-ag, wbkf, and wkdd  <cit> .
the present study includes an analysis of some of these genes and the results not only show the existence of specific markers but, more important, they also improve our understanding of the genetics-structure relationship in brucella lps.
concerning the o-polysaccharide, the results are relevant to interpret the variations in o-polysaccharide linkages of s brucella and add further weight to our previous finding  that the putative mannose genes in wbk are not essential for perosamine synthesis.
furthermore, they help to explain the differences existing between s and r brucella species.
despite extensive transposon mutagenesis searches, only four putative glycosyltransferase genes have been implicated in n-formylperosamine polymerization in brucella: wbka, wbke, wboa and wbob.
as mentioned above, wbka is conserved in classical brucella species  <cit> , and the results reported here show that wboa, wbob and wbke are similarly present in s b. melitensis, b. abortus, b. suis, b. pinnipedialis and b. ceti.
moreover, these genes displayed low polymorphism, no matter the a or m serotype.
it has to be noted that the consensus sequences of glycosyltransferases are conspicuous enough to make unlikely the existence of o-polysaccharide transferases other than wboa, wbob, wbka and wbke, and that, although the α  linkage relates to the m serotype, there is evidence showing that at least some a dominant strains generate a very small proportion  of α  linkages  <cit> .
in keeping with this, it has been observed that strain rb <dig>  generates small amounts of atypical m-type polysaccharides  <cit> .
all this evidence suggests that, rather than the presence of a α -specific transferases in the m serotype, there are subtle variations in the expression of wbob, wbka or wbke, or in the activity of the corresponding glycosyltransferases that lead to the increase in α  linkages typical of the m and a = m serotypes.
a surprising feature of the wbk is the presence of genes that are not essential for o-polysaccharide synthesis.
godfroid et al.
<cit>  analyzed the functions of the orfs between bmei <dig>  and bmei <dig>  and found that disruption of orf bmei <dig>  generated no r phenotype.
later, it was found that the genome of b. melitensis contains three putative mannose synthesis genes  adjacent to wbka.
because mannose is the direct precursor of perosamine and o-polysaccharide genes usually cluster together, monreal et al.
<cit>  proposed the names of manao-ag, manbo-ag, manco-ag for bmei <dig> to bmei <dig>  and their assignment to wbk is supported by the finding by gonzález et al.
<cit>  that disruption of orf bme <dig>  blocks o-polysaccharide synthesis.
the latter authors provided proof that at least manbo-ag, is dispensable for perosamine synthesis but also pointed out that the existence of manbcore-manccore  preclude to rule out any role for the wbk putative mannose synthesis genes since there could be internal complementation  <cit> .
all these results are fully consistent with the observation that, although manbo-ag is disrupted by is <dig> in b. pinnipedialis and b. ceti, these two species keep the s phenotype.
the wbk region has features suggestive of horizontal acquisition  <cit>  whereas manbcore  are brucella older genes necessary for the synthesis of the lps core oligosaccharide  <cit> .
accordingly, a drift to dysfunction of the wbk man genes may have been made possible by the redundancy created after horizontal acquisition of wbk, and the similarity in this regard between b. ceti and b. pinnipedialis suggests a common ancestor.
the results of this research also shed additional light on the genetic basis behind the r phenotype of b. ovis and b. canis.
previous work has shown a large deletion in b. ovis that encompasses wboa and wbob  <cit> .
the present work confirms the absence of these two putative perosaminyltraneferase genes in b. ovis, an absence that can account by itself for the lack of o-polysaccharide in this species  <cit> .
to this evidence, the present work adds the nucleotide deletion detected in b. ovis wbkf.
indeed, the frame-shift thus created predicts a very modified protein.
presumably, wbkf is involved in catalyzing the transfer of an acetylated aminosugar to undecaprenylphosphate, thus priming this carrier for o-chain polymerization.
the n-terminal region of the e. coli wbkf homologue was found to be necessary for this function  <cit>  and, therefore, it seems likely that the frame-shift in b. ovis wbkf produces a non-functional protein, thus explaining in part the r phenotype of this species.
other changes detected in several b. ovis lps genes do not have this dramatic effect.
as discussed above, the man wbk genes are dispensable and, therefore, the nucleotide substitution and frame shift detected in b. ovis manao-ag do not contribute to the r phenotype.
since disruption of manbcore generates a deep r-lps  <cit> , the presence of two more nucleotides in the sequence of b. ovis manbcore was interesting.
however, this deletion modified only the c-terminal sequence  of the protein making unlikely a change severe enough to contribute to the r phenotype.
in support of this interpretation, b. ovis r-lps is not deeply truncated like that of manbcoremutants.
moreover, the same two nucleotide addition was detected in b. suis, and it is known that a functional manbcore is required for the synthesis of s-lps in this species  <cit> .
a dna deletion of  <dig> bp.
including 3' end of wbkf and 3' end of wbkd was detected in b. canis, which might have occurred by a slipped mispairing mechanism .
it is clear that this deletion has profound consequences in the synthesis of lps.
we have discussed above the essential role of wbkf in o-polysaccharide synthesis, and wbkd seems involved in the synthesis of quinovosamine, a sugar that is possibly linking the brucella o-polysaccharide to the r-lps  <cit> .
this double mutation clearly explains the r phenotype of b. canis and is consistent with the absence of quinovosamine in this species  <cit> .
CONCLUSIONS
the analyses carried out suggest new hypothesis to study the genetics of brucella o-polysaccharide serotypes and provide evidence on both the dispensability of some wbk genes which is consistent with their horizontal acquisition.
they also confirm the essential role of wbkd and wbkf in o-polysaccharide synthesis and, at the same time, contribute to understand the r phenotype of b. ovis and b. canis.
finally, they provide several biovar and species specific markers that can be used to design the corresponding molecular typing tools.

 the members of the genus brucella are gram-negative bacteria that cause brucellosis, a zoonotic disease of great importance worldwide. currently, several brucella species are recognized. b. abortus, b. melitensis, b. suis, b. neotomae, b. ovis, and b. canis have been known for a long time and are traditionally distinguished according to their preferential host, biochemical tests and cell surface characteristics. in addition, brucella strains isolated from cetaceans and pinnipeds during the last fifteen years have been grouped into b. ceti and b. pinnipedialis,. very recently, some brucella strains have been isolated from the common vole and a new species, b. microti, proposed. b. abortus, b. melitensis and b. suis have been classically subdivided into biovars according to h2s production, co2-dependence, dye sensitivity and distribution of the a and m epitopes. however, because these tests are difficult to standardize, molecular markers have been investigated. wild type b. melitensis, b. abortus, b. suis, b. neotomae, b. ceti, b. pinnipedialis and b. microti express a smooth -type lipopolysaccharide formed by an o-polysaccharide connected to a core oligosaccharide which, in turn, is linked to lipid a, the section embedded into the outer membrane. however, both b. ovis and b. canis lack the o-polysaccharide and, accordingly, their lps is termed rough. brucella lps is of great interest not only because of these species differences but also because it is the foremost diagnostic antigen and a major virulence factor. despite this, the structure and genetics of brucella lps is only partially understood. the o-polysaccharide is a homopolymer of n-formyl-perosamine in α or in α plus α linkages, and these variations relate to the main serovars in brucella s species linkage; m dominant; or a = m ). previous studies in b. melitensis m and h have identified two genetic regions involved in o-polysaccharide synthesis and translocation. region wbo encodes two putative glycosyltransferases and region wbk contains the genes putatively involved in perosamine synthesis, its formylation and polymerization, as well as those for bactoprenol priming and o-ps translocation. in addition, wbk contains genes which may code for the enzymes that furnish mannose, the perosamine precursor. intriguingly, wbkb and manbo-ag do not generate r phenotypes upon disruption, and b. ovis and b. canis carry wbk genes despite the absence of the o-polysaccharide. much less is known on the brucella core oligosaccharide. reportedly, it contains 2-keto, 3-deoxyoctulosonic acid, mannose, glucose, glucosamine and quinovosamine but the structure is unknown. thus far, only three genes have been proved to be involved in core synthesis: pgm, manbcore and wa**. obviously, genetic analysis encompassing a variety of strains could shed light on the differences behind the phenotypes of s and r species, confirm or rule out a role for known genes, and identify differences that could serve as serovar or biovar markers. with these aims, wbke, manao-ag, manbo-ag, manco-ag, wbkf, wkdd, wboa, wbob, wa** and manbcore were analyzed for polymorphism in the classical brucella spp., b. ceti, and b. pinnipedialis.  lps genes in brucella spp. and biovars gene polymorphism in wbk wbke for all strains, the wbke pcr-amplified product displayed the same ecorv, hinfi, psti and pvuii rflp patterns. although b. melitensis 63/ biovar showed a different styi pattern, only one of eight additional b. melitensis biovar strains tested showed this styi pattern. manao-ag b. neotomae had a distinct manao-ag restriction pattern consisting of an additional avaii site. moreover, in silico analysis showed a specific profile for b. ovis consisting of a nucleotide substitution at position which modified the mana c-terminal sequence at amino acid. also, a single nucleotide deletion was detected at position 738; this frame shift leads to a change in amino acid sequence after position nucleotide sequence of pcr products from several strains confirmed the deletion in manao-ag as characteristic of b. ovis. species b. melitensis b. abortus b. suis b. ovis b. canis b. neotomae b. pinnipedialis b. ceti a atcc, american type culture collection; bccn, brucella culture collection, nouzilly, france. na: not amplified manco-ag despite the use of several endonucleases, manco-ag restriction patterns were identical in all brucella strains. therefore, no polymorphism was observed by this method. manbo-ag b. melitensis m and b. abortus tulya presented a similar manbo-ag restriction pattern, and b. melitensis biovars and showed a sau3a site absent in other strains. all b. abortus ) strains tested showed a specific pattern characterized by the absence of the ecorv site at position. b. suis biovars and b. canis and b. neotomae formed a separate group on the basis of the sau3a restriction patterns of this gene. b. ovis shared this pattern only partially because it lacked one more sau3a site. b. suis biovar strains lacked the manbo-ag sau3a site and showed an additional hinfi site in this gene. when this gene was amplified from b. ovis 63/ sequenced, and aligned with the homologous genes of b. melitensis biovar b. abortus biovar and b. suis biovar polymorphism in both sequence and length was detected. as compared to b. melitensis biovar and b. abortus biovar two more nucleotides were found at position 1265– in b. suis biovar and b. ovis which should lead to a modification of c-terminal sequence of the protein. all strains isolated from marine mammals yielded restriction manbo-ag patterns very different from those of the six classical species as well as a larger pcr product. sequencing of the pcr product of three strains revealed an is element inserted into the gene, and this insertion was confirmed by pcr in additional marine mammal strains. wbkd and wbkf the wbkd pcr product was tested with hinfi, avaii, sau3a, bgli, clai and styi, but a very low degree of dna polymorphism was observed. for b. melitensis, b. neotomae and all marine mammal strains, all strains showed the same sau3a pattern. an additional sau3a site was observed for all b. abortus, b. suis and b. ovis strains. interestingly, the b. canis product showed a reduced size of around bp and, therefore, yielded species specific restriction patterns. this result indicated the existence of a deletion in b. canis wbkd. the wbkf pcr product showed also a low degree of polymorphism when tested with ecorv, haeii, hinfi, alui, sau3a and styi. one pattern, however, was specific for b. melitensis biovar which lacked an alui site, and a distinct pattern for two b. abortus biovar and 45/ was also observed with alui site. remarkably, no amplification was obtained for b. canis, suggesting that the sequence of the wbkf-b primer corresponded to a deletion extending from the adjacent wbkd gene. in fact, when the appropriate primer was used, the wbkf pcr product showed a reduced size of about bp. to examine this point further, the wbkf-wbkd locus was amplified and sequenced in b. melitensis, b. ovis and b. canis. the sequences showed a bp deletion in b. canis extending from wbkd nucleotide to wbkf nucleotide as confirmed by the genome sequence of b. canis rm 6/. moreover, as compared to their homologs in b. melitensis, b. abortus and b. suis, gene wbkf of b. ovis showed a single nucleotide deletion at position this frame shift mutation necessarily leads to an extensive modification of cognate protein. gene polymorphism in wboa a low degree of dna polymorphism was observed in wboa. however, one pattern was specific of b. abortus since all strain testedlacked an alui site. as described above, no amplification was observed for any b. ovis strain. this confirms that absence of wboa is a b. ovis species-specific marker. polymorphism in core lps genes despite using six restriction enzymes, all brucellae displayed the same rflp pattern for the manbcore amplicon. in silico, the four genomes available showed low polymorphism. a single nucleotide deletion at position was detected in b. ovis, which should modify the c-terminal sequence of the protein. similarly, a low degree of polymorphism was observed in wa**. with the exception of b. suis biovar one psti pattern was specific of b. suis. biovar also lacked an avaii site, which could be considered as a biovar marker. with hinf a pattern was specific of b. ovis. discussion despite the high dna homology of brucellae, gene polymorphism and species- and biovar-specific markers have been consistently found. concerning outer membrane molecules, both have been found in genes of proteins but not in the lps genes examined, all of the wbk region. interestingly, these o-polysaccharide genes were found to be highly conserved not only in the classical s brucella species and biovars but also in b. ovis and b. canis, the two species that lack the o-polysaccharide. therefore, an implication of these observations is that the r phenotype of b. ovis and b. canis cannot be explained by the absence of any of those seven wbk genes. more recently, the wbk region has been extended to include wbke, manao-ag, manbo-ag, manco-ag, wbkf, and wkdd. the present study includes an analysis of some of these genes and the results not only show the existence of specific markers but, more important, they also improve our understanding of the genetics-structure relationship in brucella lps. concerning the o-polysaccharide, the results are relevant to interpret the variations in o-polysaccharide linkages of s brucella and add further weight to our previous finding that the putative mannose genes in wbk are not essential for perosamine synthesis. furthermore, they help to explain the differences existing between s and r brucella species. despite extensive transposon mutagenesis searches, only four putative glycosyltransferase genes have been implicated in n-formylperosamine polymerization in brucella: wbka, wbke, wboa and wbob. as mentioned above, wbka is conserved in classical brucella species, and the results reported here show that wboa, wbob and wbke are similarly present in s b. melitensis, b. abortus, b. suis, b. pinnipedialis and b. ceti. moreover, these genes displayed low polymorphism, no matter the a or m serotype. it has to be noted that the consensus sequences of glycosyltransferases are conspicuous enough to make unlikely the existence of o-polysaccharide transferases other than wboa, wbob, wbka and wbke, and that, although the α linkage relates to the m serotype, there is evidence showing that at least some a dominant strains generate a very small proportion of α linkages. in keeping with this, it has been observed that strain rb generates small amounts of atypical m-type polysaccharides. all this evidence suggests that, rather than the presence of a α -specific transferases in the m serotype, there are subtle variations in the expression of wbob, wbka or wbke, or in the activity of the corresponding glycosyltransferases that lead to the increase in α linkages typical of the m and a = m serotypes. a surprising feature of the wbk is the presence of genes that are not essential for o-polysaccharide synthesis. godfroid et al. analyzed the functions of the orfs between bmei and bmei and found that disruption of orf bmei generated no r phenotype. later, it was found that the genome of b. melitensis contains three putative mannose synthesis genes adjacent to wbka. because mannose is the direct precursor of perosamine and o-polysaccharide genes usually cluster together, monreal et al. proposed the names of manao-ag, manbo-ag, manco-ag for bmei to bmei and their assignment to wbk is supported by the finding by gonzález et al. that disruption of orf bme blocks o-polysaccharide synthesis. the latter authors provided proof that at least manbo-ag, is dispensable for perosamine synthesis but also pointed out that the existence of manbcore-manccore preclude to rule out any role for the wbk putative mannose synthesis genes since there could be internal complementation. all these results are fully consistent with the observation that, although manbo-ag is disrupted by is in b. pinnipedialis and b. ceti, these two species keep the s phenotype. the wbk region has features suggestive of horizontal acquisition whereas manbcore are brucella older genes necessary for the synthesis of the lps core oligosaccharide. accordingly, a drift to dysfunction of the wbk man genes may have been made possible by the redundancy created after horizontal acquisition of wbk, and the similarity in this regard between b. ceti and b. pinnipedialis suggests a common ancestor. the results of this research also shed additional light on the genetic basis behind the r phenotype of b. ovis and b. canis. previous work has shown a large deletion in b. ovis that encompasses wboa and wbob. the present work confirms the absence of these two putative perosaminyltraneferase genes in b. ovis, an absence that can account by itself for the lack of o-polysaccharide in this species. to this evidence, the present work adds the nucleotide deletion detected in b. ovis wbkf. indeed, the frame-shift thus created predicts a very modified protein. presumably, wbkf is involved in catalyzing the transfer of an acetylated aminosugar to undecaprenylphosphate, thus priming this carrier for o-chain polymerization. the n-terminal region of the e. coli wbkf homologue was found to be necessary for this function and, therefore, it seems likely that the frame-shift in b. ovis wbkf produces a non-functional protein, thus explaining in part the r phenotype of this species. other changes detected in several b. ovis lps genes do not have this dramatic effect. as discussed above, the man wbk genes are dispensable and, therefore, the nucleotide substitution and frame shift detected in b. ovis manao-ag do not contribute to the r phenotype. since disruption of manbcore generates a deep r-lps, the presence of two more nucleotides in the sequence of b. ovis manbcore was interesting. however, this deletion modified only the c-terminal sequence of the protein making unlikely a change severe enough to contribute to the r phenotype. in support of this interpretation, b. ovis r-lps is not deeply truncated like that of manbcoremutants. moreover, the same two nucleotide addition was detected in b. suis, and it is known that a functional manbcore is required for the synthesis of s-lps in this species. a dna deletion of bp. including 3' end of wbkf and 3' end of wbkd was detected in b. canis, which might have occurred by a slipped mispairing mechanism. it is clear that this deletion has profound consequences in the synthesis of lps. we have discussed above the essential role of wbkf in o-polysaccharide synthesis, and wbkd seems involved in the synthesis of quinovosamine, a sugar that is possibly linking the brucella o-polysaccharide to the r-lps. this double mutation clearly explains the r phenotype of b. canis and is consistent with the absence of quinovosamine in this species.  the analyses carried out suggest new hypothesis to study the genetics of brucella o-polysaccharide serotypes and provide evidence on both the dispensability of some wbk genes which is consistent with their horizontal acquisition. they also confirm the essential role of wbkd and wbkf in o-polysaccharide synthesis and, at the same time, contribute to understand the r phenotype of b. ovis and b. canis. finally, they provide several biovar and species specific markers that can be used to design the corresponding molecular typing tools. 
in b. pinnipedialis and b. ceti, manbo-ag carried an is confirming its dispensability for perosamine synthesis. the results define species and biovar markers, confirm the dispensability of manbo-ag for o-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth brucella species. b. ovis wbkf carried a frame-shift and b. canis had a long deletion partially encompassing both genes. b. ovis and b. canis lack the o-polysaccharide.
 the lipopolysaccharide is a major antigen and virulence factor of brucella, an important bacterial pathogen. in smooth brucellae, lipopolysaccharide is made of lipid a-core oligosaccharide and n-formylperosamine o-polysaccharide. b. ovis and b. canis lack the o-polysaccharide.  the polymorphism of o-polysaccharide genes wbke, manao-ag, manbo-ag, manco-ag, wbkf and wbkd) and wbo, and core genes manbcore and wa** was analyzed. although most genes were highly conserved, species- and biovar-specific restriction patterns were found. there were no significant differences in putative n-formylperosamyl transferase genes, suggesting that brucella a and m serotypes are not related to specific genes. in b. pinnipedialis and b. ceti, manbo-ag carried an is confirming its dispensability for perosamine synthesis. significant differences between smooth and rough species were found in wbkf and wbkd, two adjacent genes putatively related to bactoprenol priming for o-polysaccharide polymerization. b. ovis wbkf carried a frame-shift and b. canis had a long deletion partially encompassing both genes. in smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism.  the results define species and biovar markers, confirm the dispensability of manbo-ag for o-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth brucella species. 