BACKGROUND
lps is an important constituent of the outer membrane of gram-negative bacteria.
variation in lps composition can have profound consequences for these cells by potentially providing resistance against bacteriophages and antimicrobial compounds as well as facilitating evasion of the host immune system in animal pathogens.
extreme variation at lps gene clusters has been reported in animal pathogenic bacteria.
recently, eleven highly divergent gene clusters were reported to occupy an lpsspecific locus in pseudomonas aeruginosa, an opportunistic human pathogen  <cit> .
the acquisition by horizontal gene transfer of a new lps biosynthetic gene cluster in vibrio cholerae is considered as a major cause for the cholera epidemic that originated in india in  <dig>  <cit> .
in plant pathogenic bacteria, lps is an important virulence factor and mutations in the genes involved in lps production result in severe virulence deficiency  <cit> .
lps has been shown to induce resistance in plants against pathogens  <cit>  and in some recent studies, lps is found to induce expression of plant defense genes  <cit>  as well as an oxidative burst reaction in cell cultures  <cit> .
since lps recognition appears to be an important aspect of plant defense responses, variation in lps gene repertoire is to be expected within different strains of plant pathogenic bacteria.
the genus xathomonas includes a number of plant pathogenic bacteria.
two related members of this genus, xanthomonas oryzae pv.
oryzae  and xanthomonas oryzae pv.
oryzicola  cause diseases of rice  <cit> .
they exhibit different tissue specificities with xoo growing in the xylem vessels while xoor grows within the intercellular spaces of the parenchymatous tissue.
xoo causes bacterial leaf blight, the most serious bacterial disease of rice.
this disease is prevalent in many rice growing countries in asia, extending from the indian subcontinent to japan and korea.
dna fingerprinting studies using multi-locus rflp and pcr probes have indicated that there is extensive genetic diversity within xoo strains isolated from various countries  <cit> .
in india, multi-locus rflp profiling has indicated that one lineage of xoo  is widely distributed within the country.
strains within the bxo <dig> lineage cluster together at about the  <dig> % similarity level in a dendrogram.
a second group of strains is quite diverse, both at the haplotypic and pathotypic level, and clusters with the bxo <dig> group at about the 55% similarity level  <cit> .
in previous research, we have reported a  <dig>  kb region in the genome of xoo strain bxo <dig> and demonstrated that it encodes three genes that are involved in biosynthesis of lps and extracellular polysaccharide  as well as in virulence  <cit> .
all the three genes have atypical g+c content, as compared to the rest of the xoo genome.
in this study, we have completed the entire sequence of this  <dig>  kb genomic locus and indicate that it encodes three additional genes, wxod, wzt and wzm, that are postulated to be involved in lps biosynthesis and transport.
these newly described genes also have atypical g+c content and all the six genes at this locus exhibit altered codon usage pattern, as compared to other xoo genes.
we present evidence that this locus is present in many, but not all, xoo strains and that it is absent in xoor.
our results indicate that there is substantial variation at this locus among various xanthomonads.
the possible significance of these results is discussed.
RESULTS
genetic organization of a xoo lps locus
in an earlier study, a novel xoo locus was reported to be required for lps and extracellular polysaccharide  production as well as virulence.
a  <dig> kb cosmid, psd <dig>  that complements mutations in this region was isolated  <cit> .
partial sequence  of this locus indicated that the region has atypical g+c content and contains three genes which encode a predicted sugar nucleotide epimerase and two predicted glycosyl transferases.
we report here the complete  <dig>  kb sequence and genomic organization of this locus in xoo strain bxo <dig> .
the insert in the psd <dig> cosmid includes  <dig> ecori fragments .
we subcloned all the fragments into pbluescript.
based on the end sequences of the inserts in the subclones and psd <dig>  the lps locus was mapped to four of these ecori fragments .
the previously obtained sequence was found to include all of the  <dig>  kb and part of the  <dig> kb fragment and the remaining sequence of this region was obtained by sequencing the  <dig>  kb and the  <dig> kb fragment .
a total sequence of  <dig>  kb was constituted by joining  <dig>  kb of previously obtained sequence  <cit>  and  <dig>  kb of new sequence.
the  <dig>  kb sequence includes  <dig>  kb of the lps locus and some flanking regions.
the additional sequence of the lps locus encodes three putative genes which encode a predicted o-antigen acetylase, a predicted abc transporter permease and a predicted atp-binding protein and three insertion sequence  elements.
all of the putative genes have been named as per bacterial polysaccharide genes nomenclature   <cit> .
the first three genes, wxoa , wxob and wxoc  have been described earlier.
the fourth gene is wxod and encodes a predicted  <dig> amino acids long protein.
a blast  <cit>  search reveals strong homology to acetyltransferases that are involved in lps modification and the best match is with an acetyltransferase from mesorhizobium loti .
interestingly, no homologs of this gene have been reported in any other xanthomonad.
the fifth gene, wzt, encodes a predicted  <dig> amino acid long protein.
a blast search reveals homology to functions involved in lps transport.
the best match is with the atpase component of an abc-type polysaccharide transport system from burkholderia fungorum .
the sixth gene, wzm, encodes a predicted  <dig> amino acid long protein which is homologous to integral membrane protein components of abc transporter systems that are involved in lps transport.
the best match is with a permease component of the abc-type polysaccharide export system from pseudomonas fluorescens pfo- <dig> .
the start codon of wzt overlaps with the stop codon of wzm.
homologs of wzt and wzm are typically present in many lps gene clusters.
interestingly, two complete insertion sequence  elements  and one truncated is element  interrupt this cluster between the genes, wxod and wzt.
isxo <dig> is a novel  <dig> bp long insertion sequence and a blast search shows homology to transposase of isrso <dig> encoded by ralstonia solanacearum .
a complete copy of the is <dig> element  and a truncated copy of the is <dig> element  are also present as indicated in fig.
<dig>  the presence of is elements is a marked feature of many lps loci  <cit> .
transcriptional orientation suggests the possibility that orfs wxoa, wxob, wxoc and wxod might constitute one operon and that orfs wzm and wzt might be transcribed together.
the overlap between the start codon of wzt and the stop codon of wzm also suggests that these two genes are co-transcribed.
the lps locus is flanked by metb, which encodes a predicted cystathionine gamma lyase, and etfa which encodes a predicted electron transport flavoprotein.
the genome sequences of xanthomonas campestris pv.
campestris  and xanthomonas axonopodis pv.
citri  have been obtained  <cit> .
the xoo metb gene  exhibits within the sequenced region, 91% and 88% nucleotide identity to metb genes of xac  and xcc , respectively.
the xoo etfa gene  exhibits within the sequenced region, 93% and 91% nucleotide sequence identity, respectively, with etfa genes in xac  and xcc .
interestingly, the lps biosynthetic gene cluster of xcc, which comprises fifteen genes, is also located between the metb and etfa genes  <cit> .
in xac, this gene cluster is missing at this locus and is replaced by a set of fourteen genes, several of which are homologous to functions involved in lps synthesis and transport.
the gene clusters present at this locus in xcc, xac and xoo have distinct nucleotide sequences, gene numbers  and gene organization.
the xoo lps cluster is a genomic island
a) atypical g+c content
the average g+c content of xoo and other xanthomonads is estimated to be around 65%  <cit> , while the average g+c content of the lps locus is  <dig> %  .
the variation is much more marked among the genes, from as low as  <dig> %  to  <dig> % .
atypical g+c content is a characteristic feature of "genomic islands" that are believed to be acquired by horizontal gene transfer.
the transposase genes encoded by isxo <dig> and is <dig> have a g+c content that is >61%, a value which is typical for the genomes of xoo and other xanthomonads.
the g+c content of metb and etfa genes that flank the genomic island have g+c content of  <dig> % and 61% respectively  which is typical of the xoo genome.
b) altered codon usage
an additional hallmark of a genomic island is the altered codon usage.
here we present a simple and graphical way of calculating and representing the codon usage differences and refer to it as codon usage pattern or cup .
eight aminoacids, i.e., glycine, valine, threonine, leucine, arginine, serine, proline and alanine, were selected to study cup because they have atleast four synonymous codons.
the percentage of synonymous codons that end with g or c was calculated for each aminoacid and gene.
this analysis was conducted for six genes of the lps island and six genes from elsewhere in the xoo genome .
we show that cup of the genes present in the genomic island is dramatically different from the typical xoo genes .
the %g+c at third codon position of synonymous codons for amino acid glycine is only  <dig>  % for genes present in the lps locus, while it is  <dig> % in case of xoo genes that are located elsewhere in the genome.
similarly, for amino acids valine, alanine, threonine, serine, arginine, leucine and proline the values are  <dig> ,  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  % respectively for genes at the lps locus, while the values are  <dig>   <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  % for the respective aminoacids in case of the typical xoo genes.
altered codon usage is a characteristic feature of horizontally acquired genes and cup clearly indicates that the xoo lps cluster is a genomic island .
the lps locus is present in the genomes of many, but not all, xoo strains
the presence of the genomic island in different xoo strains was assessed by pcr using gene specific primers, for all the six lps genes, as described in the methods.
the list of strains used in the study is given in the table  <dig> and the list of gene specific primers is given in table  <dig>  in order to confirm that the genomic island is present at the same genomic location in all strains, pcr was also performed using two primer pairs that are designed to amplify fragments from metb to wxoa and wzm to etfa, respectively.
the analysis included nine indian xoo strains representing different geographic locations and the bxo <dig> and non bxo <dig> groups.
the list also includes twelve xoo strains from different asian countries and a xoor strain, bxor <dig>  from india.
our study revealed that the genomic island is present in the majority  of xoo strains that we have examined from india .
four bxo <dig> group strains  and three of the non-bxo <dig> strains  have the genomic island.
the genomic island is also present in two strains each from china, malaysia, indonesia, philippines, korea and one strain from nepal .
the lps locus is present, in all these strains, between the metb and etfa genes.
interestingly, we find that the genomic island is not present  in the genomes of xoo strains bxo <dig> and nepal <dig>  as well as the xoor strain, bxori.
the results obtained with the probes directed against the wxoa gene are presented but similar results were obtained using probes that are specific for the other five genes.
the blots used above were reprobed as a positive control with a metb specific probe and the results gave an expected size band in bxo <dig> , and different sized bands in bxo <dig> , nepal <dig>  and bxor <dig>  indicating that the metb gene is present but located in different ecori fragments .
bxo <dig> and nepal  <dig> have sequences related to xac at the lps locus
what are the sequences present at this genomic location in the xoo strains that lack the lps locus?
thermal asymmetric interlaced  pcr is an efficient technique for isolation of target dna segments adjacent to known sequences  <cit> .
tail-pcr and sequencing using primers directed against the conserved flanking metb and etfa genes suggests that sequences which are significantly similar to the xac lps gene cluster are present at this genomic location in both of these strains.
next to metb, a wzm homolog is present in bxo <dig>  and xac with  <dig> % identity at nucleotide level within the sequenced region.
next to etfa, a putative integral membrane protein encoding gene is present in both bxo <dig>  and xac with  <dig> % identity at nucleotide level within the sequenced region.
the bxo <dig> and nepal <dig> strains exhibit 100% nucleotide sequence identity within the sequenced region.
tail- pcr analysis of the xoor strain indicates that it has a hybrid lps gene cluster.
next to metb, a unique wzm gene is located  which exhibits  <dig> % nucleotide identity to wzm gene of pseudomonas syringae pv.
tomato strain dc <dig> .
next to etfa, a putative inner membrane protein encoding gene is located  which exhibits 97% and 92% nucleotide sequence identity, respectively, with similarly located genes in bxo <dig> and xac.
because the bxo <dig> and nepal <dig> strains have different sequences at the lps locus, as compared to other xoo strains, we inoculated these strains along with appropriate controls onto leaves of the susceptible rice cultivar taichung native- <dig>  we find that bxo <dig> and nepal <dig> strains are able to cause typical bacterial leaf blight disease symptoms that are indistinguishable from those elicited by other xoo strains .
presence of inverse repeats at the 3' ends of metb and etfa genes that flank the lps locus
we have performed an alignment using blast <dig>  <cit>  of the nucleotide sequences derived from the metb and etfa genes in bxo <dig> and bxo <dig>  the homology breakpoints appear to localise to the 3' regions of metb and etfa genes, exactly  <dig> bp upstream of their respective stop codons.
upto the break points, within the sequenced region at either end of the lps locus, the nucleotide sequence is identical in bxo <dig>  bxo <dig> and nepal <dig>  the dna sequence immediately preceding the break points was examined manually for presence of direct or inverse repeats.
interestingly, we could find three inverted repeats  within the 3' regions of metb and etfa near the homology breakpoints between bxo <dig> and bxo <dig> .
the first repeat is the smallest one  and the third repeat is the largest .
the second repeat is  <dig> bp long and is  <dig> bp from the first repeat on the metb side and  <dig> bp from the first repeat on the etfa side.
the distance between the second and third repeats is  <dig> bp in metb and etfa.
we also found similarly located inverse repeats in the metb and etfa genes of xac, xcc and xoor.
a consensus sequence of the repeats was derived  by scoring a nucleotide if it is present in a majority of repeats.
relationship between bxo <dig> and nepal  <dig> strains
the tail pcr results indicate that the bxo <dig> and nepal <dig> strains have identical sequences in place of the bxo <dig> lps locus.
as both the strains are from the indian subcontinent, there is the possibility that these are identical/nearly identical to each other.
we therefore performed dna fingerprinting analysis of the bxo <dig> and nepal <dig> strains using the is <dig> insertion element as a probe.
this probe is highly informative and can clearly differentiate the bxo <dig> and non bxo <dig> group of strains in india  <cit> .
the following strains were also included in the analysis: bxo <dig>  three non bxo <dig> group strains  and bxori.
the hybridisation pattern revealed that bxo <dig> and nepal <dig> are quite distinct from each other .
we could score  <dig> unique bands and the data generated were used to calculate pairwise similarity coefficients and cluster analysis was performed to generate a dendrogram using upgma .
the similarity coefficient between bxo <dig> and nepal <dig> is only 56%.
the dendrogram  indicates that bxo <dig> clusters with bxo#s  <dig>   <dig> and  <dig> at about the 58% similarity level while nepal <dig> clusters with all these four strains at about the 53% similarity level.
all of the xoo strains cluster with each other at about the 51% similarity level.
although the bootstrap values for these clusters are low, it is clear that the bxo <dig> and nepal <dig> strains are not closely related to each other.
as expected for an outgroup strain, bxor <dig> clusters with xoo strains at the 29% similarity level and the bootstrap value for this cluster is a high  <dig> %.
discussion
we report here the complete sequence and genomic organization of the lps locus in the bxo <dig> strain of xoo.
three of the genes in this locus i.e., wxoa, wxob and wxoc were shown in an earlier study to be required for lipopolysaccharide production and virulence  <cit> .
the predicted proteins encoded by the three new genes i.e., wxod, wzt and wzm described in the present study are homologous to functions involved in lipopolysaccharide modification and transport.
the wxod gene encodes a predicted o-antigen acetylase which is homologous to similar functions encoded in phage genomes and other bacteria.
o-antigen is the most variable part of lps.
acetylation of o-antigen is shown to confer resistance to anitimicrobial peptides in proteus mirabilis  <cit>  and determines serotype in many bacterial pathogens  <cit> .
the other two genes, wzm and wzt, are typically present in most lps gene clusters  <cit>  as tandem genes and encode functions involved in lps transport.
the wzm and wzt genes of bxo <dig> have overlapping orfs, an arrangement that is also seen in wzm and wzt genes of the lps loci in other bacteria including xac.
is elements are frequently found interrupting many lps loci  <cit>  and in bxo <dig>  three is elements interrupt the gene cluster between wxod and wzt genes.
the complete genome sequences of more than  <dig> bacteria are now available  <cit>  and studies have revealed the presence of dna segments with g+c content and codon usage different from the rest of the genome.
these regions are referred to as genomic islands and are believed to be acquired by horizontal gene transfer  <cit> .
another feature of genomic islands is their absence from the genomes of closely related strains.
our study clearly indicates that the lps locus of xoo strain bxo <dig> fulfils all of the above criterion and constitutes a genomic island.
the g+c content of this lps locus, excluding the is elements, is 50%.
the transposases encoded by isxo <dig> and is <dig> have a g+c content that is >61%.
this value, which is typical for the genomes of xoo and other xanthomonads  <cit> , suggests the possibility that these elements have transposed into the lps locus after it's transfer into the xoo genome.
the presence of this genomic island in xoo strains that are distributed across a vast segment of the asian continent suggests that it was introduced into the xoo genome early in the evolution of this pathogen.
the bxo <dig> and nepal <dig> strains do not have the lps locus that is present in the other xoo strains.
the related xanthomonad, xoor, also has an lps locus that is different from the bxo <dig> lps locus.
also, different gene clusters are present at this locus in xac and xcc .
this indicates that multiple hgt events have occurred at this locus among xanthomonads.
one hgt event occurred early in  the evolution of the xoo pathogen.
this led to the introduction of the genomic island described in figure  <dig>  two separate hgt events are likely to have occurred in the lineages that gave rise to bxo <dig> and nepal <dig> xoo strains.
this is inferred from the observation that bxo <dig> and nepal <dig> are quite unrelated in their genomic background.
another hgt can be inferred to have occurred in the xoor strain wherein sequences that are most closely related to pseudomonas syringae pv.
tomato have been introduced at one end of the lps cluster.
at least one more hgt has occurred to differentiate the lps gene clusters in xcc and xac.
the presence of invert repeats in the regions that flank the lps locus is likely to be significant.
the presence of these repeats in the metb and etfa genes is especially striking as both genes encode completely different functions.
the location of the repeats flanking the xoo lps locus suggests that they might be involved in promoting recombination during hgt and/or gene regulation.
a short inverted repeat sequence  flanking the lipopolysaccharide gene cluster has been reported in mycobacterium avium subsp.
paratuberculosis  <cit> .
another conserved sequence, called jumpstart has been found located in intergenic regions upstream of polysaccharide biosynthetic gene clusters in several animal pathogenic bacteria like escherichia coli strain k <dig>  vibrio cholera, etc.
this sequence was implicated to be involved in gene regulation and has also been suggested to have a role in recombination  <cit> .
as lps is highly immunogenic, lps loci of animal pathogenic bacteria are under intense host selection and extreme variation is reported in lps specific gene clusters  <cit> .
the observation that the two xoo strains have different lps gene clusters suggests that the plant pathogenic bacteria are also under selection to vary their lps.
alterations in lps composition might result in resistance against predators like bacteriophages  <cit>  or reduced susceptibility to certain anti-microbial compounds  <cit>  in the host/environment.
most importantly, it might help in evasion of the host defense response.
CONCLUSIONS
these results provide, for the first time, evidence for substantial variation in lps biosynthetic gene clusters within different strains of a plant pathogenic bacterium.
the results also indicate that multiple hgt events have occurred at this locus in various xanthomonads and provide a new parallel in the mechanisms that plant and animal pathogenic bacteria can employ to generate variability in cell surface molecules.

 lps is an important constituent of the outer membrane of gram-negative bacteria. variation in lps composition can have profound consequences for these cells by potentially providing resistance against bacteriophages and antimicrobial compounds as well as facilitating evasion of the host immune system in animal pathogens. extreme variation at lps gene clusters has been reported in animal pathogenic bacteria. recently, eleven highly divergent gene clusters were reported to occupy an lpsspecific locus in pseudomonas aeruginosa, an opportunistic human pathogen. the acquisition by horizontal gene transfer of a new lps biosynthetic gene cluster in vibrio cholerae is considered as a major cause for the cholera epidemic that originated in india in. in plant pathogenic bacteria, lps is an important virulence factor and mutations in the genes involved in lps production result in severe virulence deficiency. lps has been shown to induce resistance in plants against pathogens and in some recent studies, lps is found to induce expression of plant defense genes as well as an oxidative burst reaction in cell cultures. since lps recognition appears to be an important aspect of plant defense responses, variation in lps gene repertoire is to be expected within different strains of plant pathogenic bacteria. the genus xathomonas includes a number of plant pathogenic bacteria. two related members of this genus, xanthomonas oryzae pv. oryzae and xanthomonas oryzae pv. oryzicola cause diseases of rice. they exhibit different tissue specificities with xoo growing in the xylem vessels while xoor grows within the intercellular spaces of the parenchymatous tissue. xoo causes bacterial leaf blight, the most serious bacterial disease of rice. this disease is prevalent in many rice growing countries in asia, extending from the indian subcontinent to japan and korea. dna fingerprinting studies using multi-locus rflp and pcr probes have indicated that there is extensive genetic diversity within xoo strains isolated from various countries. in india, multi-locus rflp profiling has indicated that one lineage of xoo is widely distributed within the country. strains within the bxo lineage cluster together at about the % similarity level in a dendrogram. a second group of strains is quite diverse, both at the haplotypic and pathotypic level, and clusters with the bxo group at about the 55% similarity level. in previous research, we have reported a kb region in the genome of xoo strain bxo and demonstrated that it encodes three genes that are involved in biosynthesis of lps and extracellular polysaccharide as well as in virulence. all the three genes have atypical g+c content, as compared to the rest of the xoo genome. in this study, we have completed the entire sequence of this kb genomic locus and indicate that it encodes three additional genes, wxod, wzt and wzm, that are postulated to be involved in lps biosynthesis and transport. these newly described genes also have atypical g+c content and all the six genes at this locus exhibit altered codon usage pattern, as compared to other xoo genes. we present evidence that this locus is present in many, but not all, xoo strains and that it is absent in xoor. our results indicate that there is substantial variation at this locus among various xanthomonads. the possible significance of these results is discussed.  genetic organization of a xoo lps locus in an earlier study, a novel xoo locus was reported to be required for lps and extracellular polysaccharide production as well as virulence. a kb cosmid, psd that complements mutations in this region was isolated. partial sequence of this locus indicated that the region has atypical g+c content and contains three genes which encode a predicted sugar nucleotide epimerase and two predicted glycosyl transferases. we report here the complete kb sequence and genomic organization of this locus in xoo strain bxo. the insert in the psd cosmid includes ecori fragments. we subcloned all the fragments into pbluescript. based on the end sequences of the inserts in the subclones and psd the lps locus was mapped to four of these ecori fragments. the previously obtained sequence was found to include all of the kb and part of the kb fragment and the remaining sequence of this region was obtained by sequencing the kb and the kb fragment. a total sequence of kb was constituted by joining kb of previously obtained sequence and kb of new sequence. the kb sequence includes kb of the lps locus and some flanking regions. the additional sequence of the lps locus encodes three putative genes which encode a predicted o-antigen acetylase, a predicted abc transporter permease and a predicted atp-binding protein and three insertion sequence elements. all of the putative genes have been named as per bacterial polysaccharide genes nomenclature. the first three genes, wxoa, wxob and wxoc have been described earlier. the fourth gene is wxod and encodes a predicted amino acids long protein. a blast search reveals strong homology to acetyltransferases that are involved in lps modification and the best match is with an acetyltransferase from mesorhizobium loti. interestingly, no homologs of this gene have been reported in any other xanthomonad. the fifth gene, wzt, encodes a predicted amino acid long protein. a blast search reveals homology to functions involved in lps transport. the best match is with the atpase component of an abc-type polysaccharide transport system from burkholderia fungorum. the sixth gene, wzm, encodes a predicted amino acid long protein which is homologous to integral membrane protein components of abc transporter systems that are involved in lps transport. the best match is with a permease component of the abc-type polysaccharide export system from pseudomonas fluorescens pfo-. the start codon of wzt overlaps with the stop codon of wzm. homologs of wzt and wzm are typically present in many lps gene clusters. interestingly, two complete insertion sequence elements and one truncated is element interrupt this cluster between the genes, wxod and wzt. isxo is a novel bp long insertion sequence and a blast search shows homology to transposase of isrso encoded by ralstonia solanacearum. a complete copy of the is element and a truncated copy of the is element are also present as indicated in fig. the presence of is elements is a marked feature of many lps loci. transcriptional orientation suggests the possibility that orfs wxoa, wxob, wxoc and wxod might constitute one operon and that orfs wzm and wzt might be transcribed together. the overlap between the start codon of wzt and the stop codon of wzm also suggests that these two genes are co-transcribed. the lps locus is flanked by metb, which encodes a predicted cystathionine gamma lyase, and etfa which encodes a predicted electron transport flavoprotein. the genome sequences of xanthomonas campestris pv. campestris and xanthomonas axonopodis pv. citri have been obtained. the xoo metb gene exhibits within the sequenced region, 91% and 88% nucleotide identity to metb genes of xac and xcc, respectively. the xoo etfa gene exhibits within the sequenced region, 93% and 91% nucleotide sequence identity, respectively, with etfa genes in xac and xcc. interestingly, the lps biosynthetic gene cluster of xcc, which comprises fifteen genes, is also located between the metb and etfa genes. in xac, this gene cluster is missing at this locus and is replaced by a set of fourteen genes, several of which are homologous to functions involved in lps synthesis and transport. the gene clusters present at this locus in xcc, xac and xoo have distinct nucleotide sequences, gene numbers and gene organization. the xoo lps cluster is a genomic island a) atypical g+c content the average g+c content of xoo and other xanthomonads is estimated to be around 65%, while the average g+c content of the lps locus is %. the variation is much more marked among the genes, from as low as % to %. atypical g+c content is a characteristic feature of "genomic islands" that are believed to be acquired by horizontal gene transfer. the transposase genes encoded by isxo and is have a g+c content that is >61%, a value which is typical for the genomes of xoo and other xanthomonads. the g+c content of metb and etfa genes that flank the genomic island have g+c content of % and 61% respectively which is typical of the xoo genome. b) altered codon usage an additional hallmark of a genomic island is the altered codon usage. here we present a simple and graphical way of calculating and representing the codon usage differences and refer to it as codon usage pattern or cup. eight aminoacids, i.e., glycine, valine, threonine, leucine, arginine, serine, proline and alanine, were selected to study cup because they have atleast four synonymous codons. the percentage of synonymous codons that end with g or c was calculated for each aminoacid and gene. this analysis was conducted for six genes of the lps island and six genes from elsewhere in the xoo genome. we show that cup of the genes present in the genomic island is dramatically different from the typical xoo genes. the %g+c at third codon position of synonymous codons for amino acid glycine is only % for genes present in the lps locus, while it is % in case of xoo genes that are located elsewhere in the genome. similarly, for amino acids valine, alanine, threonine, serine, arginine, leucine and proline the values are, and % respectively for genes at the lps locus, while the values are,,,, and % for the respective aminoacids in case of the typical xoo genes. altered codon usage is a characteristic feature of horizontally acquired genes and cup clearly indicates that the xoo lps cluster is a genomic island. the lps locus is present in the genomes of many, but not all, xoo strains the presence of the genomic island in different xoo strains was assessed by pcr using gene specific primers, for all the six lps genes, as described in the methods. the list of strains used in the study is given in the table and the list of gene specific primers is given in table in order to confirm that the genomic island is present at the same genomic location in all strains, pcr was also performed using two primer pairs that are designed to amplify fragments from metb to wxoa and wzm to etfa, respectively. the analysis included nine indian xoo strains representing different geographic locations and the bxo and non bxo groups. the list also includes twelve xoo strains from different asian countries and a xoor strain, bxor from india. our study revealed that the genomic island is present in the majority of xoo strains that we have examined from india. four bxo group strains and three of the non-bxo strains have the genomic island. the genomic island is also present in two strains each from china, malaysia, indonesia, philippines, korea and one strain from nepal. the lps locus is present, in all these strains, between the metb and etfa genes. interestingly, we find that the genomic island is not present in the genomes of xoo strains bxo and nepal as well as the xoor strain, bxori. the results obtained with the probes directed against the wxoa gene are presented but similar results were obtained using probes that are specific for the other five genes. the blots used above were reprobed as a positive control with a metb specific probe and the results gave an expected size band in bxo, and different sized bands in bxo, nepal and bxor indicating that the metb gene is present but located in different ecori fragments. bxo and nepal have sequences related to xac at the lps locus what are the sequences present at this genomic location in the xoo strains that lack the lps locus? thermal asymmetric interlaced pcr is an efficient technique for isolation of target dna segments adjacent to known sequences. tail-pcr and sequencing using primers directed against the conserved flanking metb and etfa genes suggests that sequences which are significantly similar to the xac lps gene cluster are present at this genomic location in both of these strains. next to metb, a wzm homolog is present in bxo and xac with % identity at nucleotide level within the sequenced region. next to etfa, a putative integral membrane protein encoding gene is present in both bxo and xac with % identity at nucleotide level within the sequenced region. the bxo and nepal strains exhibit 100% nucleotide sequence identity within the sequenced region. tail- pcr analysis of the xoor strain indicates that it has a hybrid lps gene cluster. next to metb, a unique wzm gene is located which exhibits % nucleotide identity to wzm gene of pseudomonas syringae pv. tomato strain dc. next to etfa, a putative inner membrane protein encoding gene is located which exhibits 97% and 92% nucleotide sequence identity, respectively, with similarly located genes in bxo and xac. because the bxo and nepal strains have different sequences at the lps locus, as compared to other xoo strains, we inoculated these strains along with appropriate controls onto leaves of the susceptible rice cultivar taichung native- we find that bxo and nepal strains are able to cause typical bacterial leaf blight disease symptoms that are indistinguishable from those elicited by other xoo strains. presence of inverse repeats at the 3' ends of metb and etfa genes that flank the lps locus we have performed an alignment using blast of the nucleotide sequences derived from the metb and etfa genes in bxo and bxo the homology breakpoints appear to localise to the 3' regions of metb and etfa genes, exactly bp upstream of their respective stop codons. upto the break points, within the sequenced region at either end of the lps locus, the nucleotide sequence is identical in bxo bxo and nepal the dna sequence immediately preceding the break points was examined manually for presence of direct or inverse repeats. interestingly, we could find three inverted repeats within the 3' regions of metb and etfa near the homology breakpoints between bxo and bxo. the first repeat is the smallest one and the third repeat is the largest. the second repeat is bp long and is bp from the first repeat on the metb side and bp from the first repeat on the etfa side. the distance between the second and third repeats is bp in metb and etfa. we also found similarly located inverse repeats in the metb and etfa genes of xac, xcc and xoor. a consensus sequence of the repeats was derived by scoring a nucleotide if it is present in a majority of repeats. relationship between bxo and nepal strains the tail pcr results indicate that the bxo and nepal strains have identical sequences in place of the bxo lps locus. as both the strains are from the indian subcontinent, there is the possibility that these are identical/nearly identical to each other. we therefore performed dna fingerprinting analysis of the bxo and nepal strains using the is insertion element as a probe. this probe is highly informative and can clearly differentiate the bxo and non bxo group of strains in india. the following strains were also included in the analysis: bxo three non bxo group strains and bxori. the hybridisation pattern revealed that bxo and nepal are quite distinct from each other. we could score unique bands and the data generated were used to calculate pairwise similarity coefficients and cluster analysis was performed to generate a dendrogram using upgma. the similarity coefficient between bxo and nepal is only 56%. the dendrogram indicates that bxo clusters with bxo#s and at about the 58% similarity level while nepal clusters with all these four strains at about the 53% similarity level. all of the xoo strains cluster with each other at about the 51% similarity level. although the bootstrap values for these clusters are low, it is clear that the bxo and nepal strains are not closely related to each other. as expected for an outgroup strain, bxor clusters with xoo strains at the 29% similarity level and the bootstrap value for this cluster is a high %. discussion we report here the complete sequence and genomic organization of the lps locus in the bxo strain of xoo. three of the genes in this locus i.e., wxoa, wxob and wxoc were shown in an earlier study to be required for lipopolysaccharide production and virulence. the predicted proteins encoded by the three new genes i.e., wxod, wzt and wzm described in the present study are homologous to functions involved in lipopolysaccharide modification and transport. the wxod gene encodes a predicted o-antigen acetylase which is homologous to similar functions encoded in phage genomes and other bacteria. o-antigen is the most variable part of lps. acetylation of o-antigen is shown to confer resistance to anitimicrobial peptides in proteus mirabilis and determines serotype in many bacterial pathogens. the other two genes, wzm and wzt, are typically present in most lps gene clusters as tandem genes and encode functions involved in lps transport. the wzm and wzt genes of bxo have overlapping orfs, an arrangement that is also seen in wzm and wzt genes of the lps loci in other bacteria including xac. is elements are frequently found interrupting many lps loci and in bxo three is elements interrupt the gene cluster between wxod and wzt genes. the complete genome sequences of more than bacteria are now available and studies have revealed the presence of dna segments with g+c content and codon usage different from the rest of the genome. these regions are referred to as genomic islands and are believed to be acquired by horizontal gene transfer. another feature of genomic islands is their absence from the genomes of closely related strains. our study clearly indicates that the lps locus of xoo strain bxo fulfils all of the above criterion and constitutes a genomic island. the g+c content of this lps locus, excluding the is elements, is 50%. the transposases encoded by isxo and is have a g+c content that is >61%. this value, which is typical for the genomes of xoo and other xanthomonads, suggests the possibility that these elements have transposed into the lps locus after it's transfer into the xoo genome. the presence of this genomic island in xoo strains that are distributed across a vast segment of the asian continent suggests that it was introduced into the xoo genome early in the evolution of this pathogen. the bxo and nepal strains do not have the lps locus that is present in the other xoo strains. the related xanthomonad, xoor, also has an lps locus that is different from the bxo lps locus. also, different gene clusters are present at this locus in xac and xcc. this indicates that multiple hgt events have occurred at this locus among xanthomonads. one hgt event occurred early in the evolution of the xoo pathogen. this led to the introduction of the genomic island described in figure two separate hgt events are likely to have occurred in the lineages that gave rise to bxo and nepal xoo strains. this is inferred from the observation that bxo and nepal are quite unrelated in their genomic background. another hgt can be inferred to have occurred in the xoor strain wherein sequences that are most closely related to pseudomonas syringae pv. tomato have been introduced at one end of the lps cluster. at least one more hgt has occurred to differentiate the lps gene clusters in xcc and xac. the presence of invert repeats in the regions that flank the lps locus is likely to be significant. the presence of these repeats in the metb and etfa genes is especially striking as both genes encode completely different functions. the location of the repeats flanking the xoo lps locus suggests that they might be involved in promoting recombination during hgt and/or gene regulation. a short inverted repeat sequence flanking the lipopolysaccharide gene cluster has been reported in mycobacterium avium subsp. paratuberculosis. another conserved sequence, called jumpstart has been found located in intergenic regions upstream of polysaccharide biosynthetic gene clusters in several animal pathogenic bacteria like escherichia coli strain k vibrio cholera, etc. this sequence was implicated to be involved in gene regulation and has also been suggested to have a role in recombination. as lps is highly immunogenic, lps loci of animal pathogenic bacteria are under intense host selection and extreme variation is reported in lps specific gene clusters. the observation that the two xoo strains have different lps gene clusters suggests that the plant pathogenic bacteria are also under selection to vary their lps. alterations in lps composition might result in resistance against predators like bacteriophages or reduced susceptibility to certain anti-microbial compounds in the host/environment. most importantly, it might help in evasion of the host defense response.  these results provide, for the first time, evidence for substantial variation in lps biosynthetic gene clusters within different strains of a plant pathogenic bacterium. the results also indicate that multiple hgt events have occurred at this locus in various xanthomonads and provide a new parallel in the mechanisms that plant and animal pathogenic bacteria can employ to generate variability in cell surface molecules. 
in animal pathogenic bacteria, horizontal gene transfer events have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide. all six open reading frames have atypical g+c content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. we report here the complete sequence of a kb virulence locus of xanthomonas oryzae pv. the genomic island is present in a number of xoo strains from india and other asian countries but is not present in two strains, one from india and another from nepal as well as the closely related rice pathogen, xanthomonas oryzae pv. since lps is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. as a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. although lps has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium. our results indicate that multiple hgt events have occurred at this locus in the xanthomonad group of plant pathogens. this is the first report of hypervariation at an lps locus between different strains of a plant pathogenic bacterium. tail-pcr analysis indicates that sequences related to xac are present at the lps locus in both bxo and nepal the xoor strain has a hybrid lps gene cluster, with sequences at the metb and etfa ends, being most closely related to sequences from xac and the tomato pathogen, pseudomonas syringae pv. the lps locus is flanked by highly conserved genes, metb and etfa, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. interestingly, two different sets of lps genes are present at this locus in the plant pathogens, xanthomonas campestris pv. oryzae encoding six genes whose products are homologous to functions involved in lps biosynthesis and transport.
 in animal pathogenic bacteria, horizontal gene transfer events have been frequently observed in genomic regions that encode functions involved in biosynthesis of the outer membrane located lipopolysaccharide. as a result, different strains of the same pathogen can have substantially different lps biosynthetic gene clusters. since lps is highly antigenic, the variation at lps loci is attributed to be of advantage in evading the host immune system. although lps has been suggested as a potentiator of plant defense responses, interstrain variation at lps biosynthetic gene clusters has not been reported for any plant pathogenic bacterium.  we report here the complete sequence of a kb virulence locus of xanthomonas oryzae pv. oryzae encoding six genes whose products are homologous to functions involved in lps biosynthesis and transport. all six open reading frames have atypical g+c content and altered codon usage, which are the hallmarks of genomic islands that are acquired by horizontal gene transfer. the lps locus is flanked by highly conserved genes, metb and etfa, respectively encoding cystathionine gamma lyase and electron transport flavoprotein. interestingly, two different sets of lps genes are present at this locus in the plant pathogens, xanthomonas campestris pv. campestris and xanthomonas axonopodis pv. citri. the genomic island is present in a number of xoo strains from india and other asian countries but is not present in two strains, one from india and another from nepal as well as the closely related rice pathogen, xanthomonas oryzae pv. oryzicola. tail-pcr analysis indicates that sequences related to xac are present at the lps locus in both bxo and nepal the xoor strain has a hybrid lps gene cluster, with sequences at the metb and etfa ends, being most closely related to sequences from xac and the tomato pathogen, pseudomonas syringae pv. tomato respectively.  this is the first report of hypervariation at an lps locus between different strains of a plant pathogenic bacterium. our results indicate that multiple hgt events have occurred at this locus in the xanthomonad group of plant pathogens. 