BACKGROUND
the xylem-inhabiting bacterium xylella fastidiosa   <cit>  has emerged, during the past decades, as an important phytopathogen, specially due to its implication with the development of pierce's disease  in north american vineyards and citrus variegated chlorosis , which affects orange trees in south america.
pd was first detected in southern california in  <dig>  when it destroyed approximately  <dig>  acres of grapes in anaheim, ca, during a 5-year outbreak of the disease .
after this devastating experience, pd remained as a minor concern to the west coast viticulture for decades until the mid-1990s, when a new insect species, the glassy-winged sharpshooter  homalodisca vitripennis was accidentally introduced into southern california and begun spreading northward.
this leafhopper, which can serve as a vector to xf, has the capacity to feed in more than  <dig> different plant species and survive winter temperatures dropping as low as 20°f  <cit> .
moreover, unlike other xf-carrying insects associated with pd in california, gwss has a much broader flight range , posing a very serious threat to the wine industry from southern and central california  <cit> .
indeed, since the first identification of gwss in the california vineyards, programs aimed at controlling the dissemination of this insect as a strategy to prevent pd outbreaks have involved more than us$  <dig> million of direct investments  <cit> .
citrus variegated chlorosis, on the other hand, was originally identified in brazil in  <dig>  during an outbreak that affected orange orchards distributed along the northern and northwestern regions of state of são paulo  <cit> , one of the most important areas of citrus production in this country, which turns out to be one of the world's leading producers of concentrated orange juice .
since its initial observation, the disease incidence increased by graft propagation of xf-infected budwood and by the action of many different sharpshooter vectors, becoming widely distributed across all citrus-growing regions in the country, where it is held responsible for damages that may reach us$  <dig> –  <dig> million per year  <cit>  .
different xf strains have also been obtained from alternative host plants across the americas and, in many cases, there seems to be a direct correlation between xf infection and the development of diseases  <cit> .
thus, xf strains are also believed to be responsible for phony peach disease , alfalfa dwarf disease, periwinkle wilt and leaf scorch diseases in plum, elm, maple, oak, sycamore and coffee  <cit> .
up to now, none of these diseases has demonstrated to be as economically damaging as either pd or cvc.
nonetheless, xf is already considered a major agronomical concern in the american continent, given the economic losses already experienced by both citrus and winegrape industries, as well as the widespread distribution of xf strains in so many economically important crops.
thus, this microorganism has been the subject of increasing attention by many research programs after the mid 1990's  <cit>  and, as a consequence of these efforts, one xf strain , associated with cvc in brazil, turned out to be the first plant bacterium to have its complete genome sequenced and annotated  <cit> .
moreover, the existence of different disease symptoms, observed in a wide range of plant hosts  <cit>  and associated with genetically distinct xf strains, has led researchers to hypothesize that total genome comparisons among these bacteria could help to uncover information regarding genes involved in the interaction with specific hosts and disease development  <cit> .
thus, sequencing efforts have been extended to other xf strains and subsequently, the genomes of two other strains , obtained from oleander and almond trees had their genomes partially sequenced and annotated  <cit> .
finally, a fourth strain, temecula- <dig>  isolated from grapevines and responsible for pd in california has also been sequenced to completion  <cit> .
thus, xf is one of the best models available to conduct functional and comparative genomic studies.
genomic sequences from these xf strains have been submitted to extensive in silico evaluations, allowing the formulation of virtual metabolomes that provided a comprehensive view of the major biochemical processes that occur in these microorganisms  <cit> .
additional information regarding the functionality of different gene products and pathogenicity mechanisms in xf have also been obtained by the evaluation of differential gene expression through microarray hybridization approaches and by the generation of gene-knockout mutants  <cit> , while genomic comparisons conducted with these four strains allowed the identification and categorization of genome-wide dna variations, as well as their influence on strain functional divergence  <cit> .
multiple alignment of chromosomal sequences identified snps and indels that could be used to estimate the relative similarity between the strains and the rates of genome evolution, which seem to be different for each individual strain  <cit> .
all unique genes have been catalogued and, since their sequences could represent strain-specific markers, primer pairs were designed against these orfs to assist in pcr-based detection of these four xf strains in the wild  <cit> .
the genomic information also established a solid base for the development of epidemiological and phylogenetic studies within the xf group, providing evidence that the bacterial species x. fastidiosa, originally characterized from  <dig> strains , constitute a significantly complex group of plant-associated bacteria  <cit> .
in spite of sharing a considerable amount of both phenotypic and genotypic similarities, as well as an overall 85% dna sequence identity, as measured from dna homology studies  <cit> , x. fastidiosa strains and strains have been shown to display significant biological variability, which was confirmed by phylogenetic analyses involving sequence comparison of seven chromosomal genes , sequencing of the ribosomal intergenic spacer , serological classification and microarray analyses of their genomic profiles  <cit> .
however, all these studies involved a relatively small number of south american xf strains, hampering a more conclusive evaluation about the biogeographical distribution, phylogenetic history, evolution and taxonomic relationships among this group of strains, which have not been as thoroughly studied as the north american strains.
a similar situation is verified when xf strains are analyzed and compared at the genomic level, since three north american strains have been submitted to genomic sequencing, as opposed to only one south american strain.
thus, it is possible that the relative lack of sequence information from other south american strains may have introduced biases to some of the conclusions drawn from recent genomic studies within the group.
this situation prompted us to conduct a comprehensive genomic survey involving a total of  <dig> south american xf strains, all obtained from infected orange trees  <cit> .
we employed a microarray-based approach to compare the genomic profiles of these bacteria with xf strain 9a5c and suppressive subtraction hybridization  to identify new genes present exclusively in the genomes of these microorganisms.
the results obtained from such analyses represent the first genome-wide evaluation regarding genome structure and composition from cvc-associated bacteria, providing additional information about the characteristics of the south american xf gene pool and its relationship with what has been found in north american strains.
RESULTS
genomic comparisons among xf-cvc strains through microarray hybridization analysis
microarray hybridization has been widely used to undertake genomic comparisons involving a great number of microorganisms  <cit>  and previous work from our group has established precise criteria to employ this methodology to the study of xf strains, with the aid of an xf 9a5c biochip  <cit> .
thus, we performed similar microarray-based comparisons with  <dig> different xf strains obtained from citrus plants .
four of these xf strains  were obtained from cvc-affected trees and are representatives of the most prevalent xf haplotypes found in sweet orange orchards across the state of são paulo, while xf strain cv <dig> was obtained from a non-symptomatic tree from the same region  <cit> .
xf strain fb <dig>  on the other hand, was obtained from a sweet orange tree that displayed symptoms of "pecosita", a disease similar to cvc that occurs in some citrus-growing regions of argentina  <cit> .
the genomic profiles obtained for these  <dig> strains are depicted in figure  <dig>  which displays a linear representation of the xf strain 9a5c chromosome , followed by orfs present in pxf <dig>  the large  <dig> kb chromosome present in xf strain 9a5c .
surprisingly, these results showed that, contrary to what had been observed during our former comparisons  <cit> , most genomic differences within the citrus strains are not associated with deleted orfs, but with elements that are present in greater copy number in the tested strains, when compared to xf strain 9a5c .
nonetheless, we were able to confirm the same distribution pattern observed before, since most duplicated orfs are not scattered throughout the genome, but grouped within mobile genetic elements, such as prophages, genomic islands  and putative genomic islands  previously observed in the genome of xf strain 9a5c  <cit> .
details regarding differences in orf composition, detected by the microarray hybridizations with all xf strains analyzed in this study, can be found in additional file  <dig>  as expected, each strain presents a unique genomic profile, which can be used to characterize all individual strains with high fidelity, as inferred from the high bootstrap values obtained from the cluster analysis shown in figure  <dig>  interestingly, the argentine strain, fb <dig>  seems to be extremely similar to xf strain 9a5c, as the genomic differences detected between these two strains are restricted to a small number of deleted orfs, distributed along the main chromosome and plasmid pxf <dig>  all other strains display significantly different profiles, characterized not only by deletions, but also by duplicated orfs.
a strong similarity is observed in the profiles obtained for xf strains 56a and  <dig> c, characterized by scattered duplication of orfs from gi <dig>  and by what seems to be complete duplications of gi <dig> and gi <dig>  several orfs from prophage xfp <dig> and gi <dig> are also missing in these two strains.
xf strain cv <dig> presents a genomic profile that closely resembles xf strains 56a and  <dig> c, except for the lack of duplications involving orfs within gi <dig>  xf strain 36f, on the other hand, displays another type of profile, in which no deletions have been detected along the structures of xfp <dig> and gi <dig> and the duplicated orfs scattered across gi <dig> and gi <dig> seem to indicate that duplication of these elements is not complete.
finally, xf strain 187b displays the most divergent genomic profile, characterized by a very large number of duplicated orfs and mobile elements.
once again, duplications seem to span practically the entire structure of gi <dig>  gi <dig> and even gi <dig>  moreover, several other duplicated regions seem to occur throughout the genome of xf strain 187b, pointing to the possible existence of other mobile genetic elements in xf.
interestingly, at least three of these regions seem to map within elements that had been previously identified as putative genomic islands   <cit> .
the two largest xf gis , which were found to be deleted in north american strains are present in all citrus strains analyzed herein and gi <dig> seems to be completely duplicated in at least four of the tested xf strains , as well as in two previously analyzed citrus xf strains, x1-b <dig> and sj  <cit> , indicating that this element seems to display intense transpositional activity among representatives of the xf group.
interestingly, gi <dig> seems to be specific to strains obtained from citrus and coffee trees from south america and may represent a sinapomorphy for the south american xf strains  <cit> .
gi <dig>  which is also duplicated in other xf strains , displays a region that is similar to the vape-containing region of gi <dig>  resembling the situation observed with the vap elements from the pathogenic bacterium dichelobacter nodosus .
identification of new orfs in the xf-cvc gene pool of citrus-associated xf strains through suppressive subtraction hybridization
since the xf biochip used in the experiments described above was based on the genome of xf strain 9a5c, the hybridization experiments can only provide information regarding genes that are present in this strain.
thus, to gather information concerning additional orfs present in the gene pool of citrus-associated strains, we employed suppressive subtraction hybridization , using dna from xf strain 9a5c as a driver, against dna from all  <dig> strains, as described in methods.
thus, a total of  <dig> ssh libraries have been constructed  and approximately  <dig>  clones have been obtained and sequenced from these libraries .
for each of the  <dig> strains, the sequenced clones have been trimmed, in order to exclude vector sequences and poor quality regions , and aligned with the aid of cap <dig>  generating  <dig>  contigs, which contained  <dig>  sequences overall.
these contigs span  <dig>  kb of xf sequences, which is equivalent to approximately  <dig> % of the genome of xf strain 9a5c .
we chose to conduct further analyses only with contigs composed by at least  <dig> different individual sequences and showed size variation from  <dig> to  <dig>  bp.
thus, a total of  <dig>  sequenced clones were excluded from further studies, since they either remained as singlets or grouped into small contigs, composed by a limited number of reads , resulting in poor quality consensus sequences.
next, the contigs were filtered against the genome of xf strain 9a5c with the aid of the software cross_match  <cit> , allowing the identification of stretches of dna that were exclusive to the tested strains.
sequencing of approximately  <dig>  ssh clones has allowed the identification of  <dig>  kb of dna sequences that are not present in the genome of xf strain 9a5c .
the rate at which novel sequences were identified, as a function of sequenced ssh clones, has been evaluated as described in methods, and judging by the inclination of the curve shown in figure  <dig>  a significant proportion of the novel sequences present in the analyzed genomes is likely to have been identified in this study.
these newly identified sequences were analyzed with genemark  <cit> , to search for individual elements present in their structure, allowing the identification of  <dig> new orfs, scattered throughout the genomes of the  <dig> strains analyzed herein .
this represents an increment of approximately  <dig> % in the number of orfs currently known to belong to the gene pool of citrus-associated xf  <cit> .
however, comparative analyses among these  <dig> newly identified orfs has shown that a large proportion of these elements  seemed to belong to different groups of orthologous proteins, present in the genomes of two or more tested strains.
thus, to reduce the redundancy of this dataset, the predicted protein sequences from these  <dig> orfs have been submitted to cluster analysis, as described in methods, resulting in  <dig> groups of orthologous elements , which are more likely to represent the actual number of new functional genes identified in the gene pool of the tested xf strains .
analyses of similarity against the genbank have then been performed with blastx, using the consensus sequences from each goe as input, which allowed the assignment of putative functions for each of these newly identified elements.
their distribution into the different functional categories originally described by simpson and co-workers is shown in figure  <dig> <cit> .
surprisingly, a relatively small proportion of such sequences has been identified as "no hit" .
another large fraction of elements  has been identified as conserved hypothetical proteins , displaying high similarity to orfs of unknown function, previously identified in the genomes of other microorganisms – particularly in other xf strains .
putative functions could be attributed to  <dig> newly identified goes  and the majority of them  are directly associated with mobile genetic elements, since we identified  <dig> new phage structural proteins,  <dig> recombinases/integrases,  <dig> elements involved with plasmid replication/stabilization and  <dig> elements that are homologous to conjugation factors belonging to the traa/trab/cag/vir families, originally described in agrobacterium tumafasciens  <cit> .
the remaining  <dig> goes,  encode proteins that are involved with several metabolic processes of the cell, including a group of six new potential virulence factors, which had not been originally identified in the genome of xf strain 9a5c.
these include two homologues for the transcription factor abrb , a new pilin gene , two lpx acetyltransferases  and a gene encoding the zonula ocludens toxin  from vibrio cholerae .
the blastx analyses also confirmed that the sequences from all  <dig> elements described above could not be found in the genome of xf strain 9a5c.
surprisingly, however, these analyses showed that a large proportion of these sequences  has already been identified in xf strains from north america – particularly in the case of xf ann- <dig>  isolated from oleander .
as shown in additional file  <dig>  the consensus sequences from  <dig> goes, showed high similarity to orfs originally described in xf strain ann- <dig>  encoding both hypothetical proteins and proteins with assigned functions.
sixteen goes showed high similarity with elements identified in the genome of xf strain dixon  and eight were most similar to sequences found in xf strain temecula- <dig> .
such an overlap with genes from the north american xf gene pool was unexpected and suggests that the list of unique orfs, recently proposed by doddapaneni and coworkers  <cit> , as potential targets for strain-specific detection of north american xf strains in the wild must be revised  <cit> .
as shown in table  <dig>  approximately  <dig> % of the orfs, previously believed to be unique to xf strain ann- <dig>  and  <dig> % of the orfs, previously believed to be unique to xf strain dixon  have also been found in the citrus-associated strains analyzed in this study.
on the other hand, since the consensus sequences from  <dig> goes returned "no hit" when compared to the ncbi databases, these elements might prove useful as targets for pcr-based detection of citrus-associated xf strains .
a as described by doddapaneni and coworkers  <cit> .
altogether, the consensus sequences from only  <dig> goes showed similarity to genes identified in microorganisms that do not belong to the xf group .
interestingly, one third of these elements  seem to be homologous to orfs identified in the genome of verminephrobacter eiseniae, a soil-inhabiting bacterium that infects nematodes  <cit> , while the remaining ones show high similarity to genes found in a great number of microorganisms, such as e. coli, geobacter metalireducens, salmonella enterica, solibacter usitatus, xanthomonas sp., burkholderia sp., chlorobium tepidum, among others.
discussion
results obtained from several lines of research, developed during the past decade, have turned the phytobacterium xylella fastidiosa into a unique model of study in the fields of both phytopathology and genomics.
first of all, this is due to the fact that several diseases, associated with many distinct bacterial strains have been characterized and some of these diseases are responsible for significant economic losses.
moreover, a significant amount of genomic information has been obtained for four different strains of this microorganism, allowing the development of both functional and comparative genomic analyses within the group.
comparisons performed with the four sequenced xf genomes led doddapaneni and coworkers to suggest that the tenecula- <dig> strain genome is the one that most likely resembles the ancestral xf genome, since it has undergone the fewest genetic changes among the four analyzed strains  <cit> .
thus, xf strain 9a5c  may have evolved from an ancestral bacterium, carrying a temecula-1-like genome, through incorporation of a series of horizontally transferred elements, such as prophages, gis and plasmids  <cit> .
this possibility is reinforced by the microarray-based comparisons undertaken in this study with the citrus-associated strains, since they provide further evidence for intense transpositional activity of mobile elements during the evolution of this subgroup of xf.
so far, most comparative studies among south american xf strains have essentially involved brazilian strains from both citrus and coffee trees.
in general, the available data supports the idea that cvc-associated bacteria found in brazil have evolved directly from xf strains that cause coffee leaf scorch , since cvc-associated xf strains have been shown to induce cls symptoms when experimentally inoculated into coffee trees  <cit> .
moreover, it is widely known that most areas in which citrus orchards are presently cultivated used to be dedicated to coffee plantations and there have been reports of cvc vectors feeding on coffee trees  <cit> .
nonetheless, the exact origin of cvc-related xf strains is still a matter of speculation, since very little research has been performed on pecosita-related xf strains.
the origin of this subgroup of xf is an important piece of information regarding the evolutionary history of citrus-associated xf strains, specially considering that pecosita has been known to occur in argentina before the first descriptions of cvc in brazil .
thus, the present study shows the first genomic-scale comparative evaluation involving a pecosita-related xf strain .
it is interesting to verify from these data that, while xf strain fb <dig>  appears to be very similar to xf strain 9a5c , significant genomic differences have been observed when these strains are compared to xf-cvc bacteria isolated in more recent years, specially regarding the activity of horizontally transferred elements.
although it is tempting to speculate from this data that the evolutionary rates among cvc- and pecosita-related strains might differ, it is clear that further phylogenetic and biogeographical studies have yet to be performed in order to shed more light into our knowledge regarding the evolutionary history of the south american xf strains, as well as their corresponding diseases.
an attempt to verify the evolutionary relationships between xf strains 9a5c, fb <dig> and the other xf strains has been performed by sequencing the 16s-23s rdna spacer region of these bacteria, but since no strain-specific mutations have been found in this sequence, such analysis turned out to be inconclusive .
as mentioned before, the fact that the overall genomic information obtained for x. fastidiosa has been mostly based on sequence information derived from north american strains, was likely to have biased to some of the conclusions obtained so far, specially considering the evolutionary complexity of these bacteria, which have been shown to carry an extremely large and active flexible gene pool  <cit> .
as expected, the ssh experiments described in this work, which constitute the first attempt to undertake a large-scale survey of the genomic composition of south american xf strains, showed that a total of  <dig> orfs, originally identified as unique to the north american strains also seem to be present in the citrus strains from south america  <cit> .
among these orfs,  <dig> had been proposed to be exclusive to xf strain ann- <dig> and  <dig> were supposed to be present only in the genome of xf strain dixon.
thus, sequencing of ssh clones from citrus-associated xf strains resulted in a reduction of approximately 19% in the list of unique genes that could be used as markers for north american strains   <cit> .
these findings reinforce the importance of gathering more information regarding the genomic composition of south american strains – including strains isolated from other hosts, such as coffee, to help in understanding the evolutionary history of the xf group and assist in the development of tools for specific identification of pathovars  <cit>  and other variants.
at this point, it is not clear whether the increasing overlap between elements found in south and north american strains are a result of ancestry, or represent lateral transfer events among strains  <cit> .
although the geographical separation of south and north american strains presents a clear barrier to lateral gene transfer events, the evolutionary history of the xf genome has been shown to be extremely dynamic, highly influenced by the activity of transpositional elements from its large flexible gene pool  <cit> .
in fact, direct evidence for the occurrence of genetic exchange between xf strains from the two continents has already been reported after analysis of one xf strain, obtained from plum trees in south america  <cit> .
regardless of its geographical origin, the genomic profile obtained for this strain clearly indicated that it descended from north american strains and was likely to have been accidentally introduced into the south american continent via infected plant material.
however, this strain carried practically all genes from pxf  <dig>  a large plasmid found exclusively in south american xf strains, which was likely acquired by conjugation events with south american strains  <cit> .
thus, given the fact that xf strains are known to infect more than  <dig> plant species – many of which are commercial crops that are constantly shipped to foreign countries  <cit>  , it is not unlikely to assume that other contamination events might have taken place, allowing the introduction of north american strains into the south american territory, and vice versa.
further conjugation events, for instance, might have introduced genes that were specific to the north american strains into the gene pool of south american strains, which could have been facilitated by the presence of such a large set of transposable elements in the xf genome and the fact that multiple strains have been shown to coexist in the same host – both in the case of infected plants, as well as insect vectors  <cit> .
the dynamics of xf genome evolution can also be deduced through evaluation of other mobile elements, particularly in the case of gi <dig>  which seems to be absent from the north american strains, but present in one or more copy numbers in south american strains, as shown in figure  <dig>  among all mobile elements identified in xf, gi <dig> is the one that best fulfills the definition of a genomic island, since it displays a higher gc content, altered codon bias, insertion at the 3'end of a trna gene  and the presence, at one end, of orfs that display high similarity to a heterodimeric integrase found in association with an insertion element from helicobacter pylori   <cit> .
more interestingly, gi <dig> may be significant for the evolution of the xf group, since all studies undertaken so far, involving a total of  <dig> different south american strains, obtained from both citrus and coffee, indicate that this element may represent a genomic sinapomorphy for the south american xf strains  <cit> .
at this point, it is not possible to determine if elements present in gi <dig> play any role in mediating adaptation of south american xf strains to their specific hosts, nor if they participate in the process plant infection and colonization.
nonetheless, this element carries a series of orfs whose products are potentially involved with host adaptation and pathogenicity, resembling the structure of pathogenicity islands  observed in other bacteria  <cit> .
for instance, gi <dig> carries a unique fimbrillin gene, represented by orf xf <dig>  fimbrillins are components of bacterial type i fimbriae, which are directly implicated in the process of attachment to different types of substrates and biofilm formation  <cit> .
novel fimbriae, composed by distinct fimbrillin genes, are believed to be important virulence determinants, allowing colonization of specific hosts and differentiation of virulent clonal groups of pathogenic bacteria, as in the case of e. coli strains, in which new fimbrillin isomorphs are also associated with horizontally transferred elements  <cit> .
interestingly, xf strains that display specificity to different host and vector species also display a variable number of fimbrilling homologues scattered throughout their genomes  <cit> .
gi <dig> has also been shown to carry toxin-associated genes, such as orf xf <dig>  which encodes a hemolysin-like protein, which is implicated in cytotoxic reactions associated with many pathogenic bacteria  <cit> , and orf xf  <dig>  involved in the synthesis of the lps fraction of gram-negative bacteria that mediates adverse reactions in both human and animal hosts during infection by pathogenic bacteria  <cit> .
in the case of phytobacteria, the o-antigen portion of lps has been shown to display a strain-specific pattern, which is believed to play a major role in host recognition during the plant colonization process  <cit> .
finally, gi <dig> also carries orf xf <dig>  which encodes the virulence-associated factor vape from dichelobacter nodosus, the causative agent of ovine footrot disease.
in this bacterium, the vap genes are present in a family of pathogenicity islands called the vap elements  <cit> .
interestingly, up to  <dig> different vap elements, varying in length and orf composition, have been mapped throughout the genome of d. nodosus and although their exact function are still unknown, their presence has been clearly shown to be associated with the virulence phenotype in this bacterium  <cit> .
curiously, as mentioned above, the vape-containing region of gi <dig> is also duplicated in xf strain 9a5c, as a part of gi <dig>  resembling the organization of vap elements in d. nodosus.
as mentioned above, additional virulence factors have now been identified in the genome of citrus-associated strains through the ssh experiments.
one of these elements encodes a new form of pilin, associated with type iv fimbriae, whose twitching motility mechanism has been shown to be of capital importance to host colonization in xf  <cit> .
this finding reinforces the importance of fimbriae for the evolutionary divergence of xf strains.
transcriptional regulators, such as the abrb activator have also been found through the ssh experiments.
this transcription factor is responsible for controlling several genes, specifically activated at the end of bacterial exponential growth phase in bacillus subtilis, and has been shown to be implicated with biofilm formation in this bacterium  <cit> .
interestingly, biofilm formation is believed to be an important virulence factor in xf during the development of both cvc and pd, since growth in biofilm is likely to participate in the process of bacterial adherence to the xylem vessels, contribute to xylem occlusion and increase bacterial survival against the oxidative burst mediated by infected plant tissues  <cit> .
two different homologues of the abrb gene have been found in  <dig> out of the  <dig> analyzed xf strains , while the new pilin gene has been found only in xf strain  <dig> c. two other potential virulence factors found in the xf-cvc gene pool seem to belong to the cyse/laca/lpxa/nodl family of acetyltransferases, which are characterized by multiple repeats of the sequence -g-x  <cit> .
in both cases, the highest similarities found for these newly identified acetyltransferases are with genes lpxa and lpxd from the north american strains ann- <dig> and temecula- <dig>  <cit> , which are involved in synthesis and modification of the o-antigen fraction of gram-negative bacteria lps.
as mentioned above, the strain-specific structure of the o-antigen has been show to be an important mediator in plant colonization by symbiotic bacteria, especially in the case of nodule formation during rhizobium-legume interactions  <cit> .
finally, the genomes of xf strains 187b and  <dig> c seem to carry a copy of the gene that encodes the zonula occludens toxin , also found in xf strain ann- <dig>  zot-like genes have been found in association with several bacterial pathogens, such as xanthomonas, vibrio and stenotrophomonas, among others  <cit>  and are likely to be laterally transferred among microorganisms through the action of filamentous phages  <cit> .
this toxin has been originally described as an important virulence factor in vibrio cholerae, and seems to be responsible for the development of severe cases of diarrhea caused by v. cholerae strains that do not carry the cholera toxin gene ctxa.
the activity of zot as an enterotoxin seems to be associated to its capacity to interfere with tight junctions of the gastrointestinal epithelium, altering its permeability to water and other substances  <cit> .
nonetheless, regardless of its recognized importance as a virulence factor in animal or human infections, there is no direct evidence that zot plays any role during plant colonization and/or pathogenicity, although this gene has also been found in the genome of xanthomonas campestris, the causative agent of black rot disease, characterized by hyper-hydration of infected plant tissues, associated with wet edged lesions, which may be a result of zot activity  <cit> .
CONCLUSIONS
the experiments described in the present study represent the first attempt to conduct microarray-based genomic comparisons and a large-scale survey of genes present in the genomes of south american xf strains.
the results from the microarray-based comparison provide further evidence concerning the intense transpositional activity of several horizontally transferred elements and reinforce previous studies regarding the importance of lateral gene transfer as a major mediator in the evolution of this important group of phytopathogens.
moreover, comparison of the microarray-based genomic profiles showed similarity between xf strains 9a5c and fb <dig>  which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms.
it is clear that a more comprehensive evaluation of both coffee- and pecosita-related strains is necessary before we can fully understand the evolutionary history of south american xf strains and their associated diseases.
the characterization of approximately  <dig>  ssh clones, from six representatives of citrus haplotypes, have now provided a more comprehensive view of the size and composition of the xf-cvc gene pool, allowing us to identify  <dig> new orfs – a number that represents an ~ <dig> % increase in our current knowledge of the south american xf gene pool.
these results allowed identification of new putative virulence factors, as well as novel potential markers for strain identification within this subgroup of phytopathogens by molecular-based approaches.
moreover, several sequences previously believed to be unique markers for north american strains have now been found in the genomes of these south american strains  <cit> .
these new data point to the necessity of revising the molecular markers currently accepted as potential targets for identification of distinct xf strains in the wild  <cit> .
finally, by evaluating the rate at which novel sequences have been identified through the ssh approach, we conclude that the complete composition of the south american xf gene pool can still be stretched, specially if this type of analysis is further extended to strains obtained from alternative hosts, such as coffee, which is known to harbor many different xf strains throughout the south american territory  <cit> .

 the xylem-inhabiting bacterium xylella fastidiosa has emerged, during the past decades, as an important phytopathogen, specially due to its implication with the development of pierce's disease in north american vineyards and citrus variegated chlorosis, which affects orange trees in south america. pd was first detected in southern california in when it destroyed approximately acres of grapes in anaheim, ca, during a 5-year outbreak of the disease. after this devastating experience, pd remained as a minor concern to the west coast viticulture for decades until the mid-1990s, when a new insect species, the glassy-winged sharpshooter homalodisca vitripennis was accidentally introduced into southern california and begun spreading northward. this leafhopper, which can serve as a vector to xf, has the capacity to feed in more than different plant species and survive winter temperatures dropping as low as 20°f. moreover, unlike other xf-carrying insects associated with pd in california, gwss has a much broader flight range, posing a very serious threat to the wine industry from southern and central california. indeed, since the first identification of gwss in the california vineyards, programs aimed at controlling the dissemination of this insect as a strategy to prevent pd outbreaks have involved more than us$ million of direct investments. citrus variegated chlorosis, on the other hand, was originally identified in brazil in during an outbreak that affected orange orchards distributed along the northern and northwestern regions of state of são paulo, one of the most important areas of citrus production in this country, which turns out to be one of the world's leading producers of concentrated orange juice. since its initial observation, the disease incidence increased by graft propagation of xf-infected budwood and by the action of many different sharpshooter vectors, becoming widely distributed across all citrus-growing regions in the country, where it is held responsible for damages that may reach us$ – million per year. different xf strains have also been obtained from alternative host plants across the americas and, in many cases, there seems to be a direct correlation between xf infection and the development of diseases. thus, xf strains are also believed to be responsible for phony peach disease, alfalfa dwarf disease, periwinkle wilt and leaf scorch diseases in plum, elm, maple, oak, sycamore and coffee. up to now, none of these diseases has demonstrated to be as economically damaging as either pd or cvc. nonetheless, xf is already considered a major agronomical concern in the american continent, given the economic losses already experienced by both citrus and winegrape industries, as well as the widespread distribution of xf strains in so many economically important crops. thus, this microorganism has been the subject of increasing attention by many research programs after the mid 1990's and, as a consequence of these efforts, one xf strain, associated with cvc in brazil, turned out to be the first plant bacterium to have its complete genome sequenced and annotated. moreover, the existence of different disease symptoms, observed in a wide range of plant hosts and associated with genetically distinct xf strains, has led researchers to hypothesize that total genome comparisons among these bacteria could help to uncover information regarding genes involved in the interaction with specific hosts and disease development. thus, sequencing efforts have been extended to other xf strains and subsequently, the genomes of two other strains, obtained from oleander and almond trees had their genomes partially sequenced and annotated. finally, a fourth strain, temecula- isolated from grapevines and responsible for pd in california has also been sequenced to completion. thus, xf is one of the best models available to conduct functional and comparative genomic studies. genomic sequences from these xf strains have been submitted to extensive in silico evaluations, allowing the formulation of virtual metabolomes that provided a comprehensive view of the major biochemical processes that occur in these microorganisms. additional information regarding the functionality of different gene products and pathogenicity mechanisms in xf have also been obtained by the evaluation of differential gene expression through microarray hybridization approaches and by the generation of gene-knockout mutants, while genomic comparisons conducted with these four strains allowed the identification and categorization of genome-wide dna variations, as well as their influence on strain functional divergence. multiple alignment of chromosomal sequences identified snps and indels that could be used to estimate the relative similarity between the strains and the rates of genome evolution, which seem to be different for each individual strain. all unique genes have been catalogued and, since their sequences could represent strain-specific markers, primer pairs were designed against these orfs to assist in pcr-based detection of these four xf strains in the wild. the genomic information also established a solid base for the development of epidemiological and phylogenetic studies within the xf group, providing evidence that the bacterial species x. fastidiosa, originally characterized from strains, constitute a significantly complex group of plant-associated bacteria. in spite of sharing a considerable amount of both phenotypic and genotypic similarities, as well as an overall 85% dna sequence identity, as measured from dna homology studies, x. fastidiosa strains and strains have been shown to display significant biological variability, which was confirmed by phylogenetic analyses involving sequence comparison of seven chromosomal genes, sequencing of the ribosomal intergenic spacer, serological classification and microarray analyses of their genomic profiles. however, all these studies involved a relatively small number of south american xf strains, hampering a more conclusive evaluation about the biogeographical distribution, phylogenetic history, evolution and taxonomic relationships among this group of strains, which have not been as thoroughly studied as the north american strains. a similar situation is verified when xf strains are analyzed and compared at the genomic level, since three north american strains have been submitted to genomic sequencing, as opposed to only one south american strain. thus, it is possible that the relative lack of sequence information from other south american strains may have introduced biases to some of the conclusions drawn from recent genomic studies within the group. this situation prompted us to conduct a comprehensive genomic survey involving a total of south american xf strains, all obtained from infected orange trees. we employed a microarray-based approach to compare the genomic profiles of these bacteria with xf strain 9a5c and suppressive subtraction hybridization to identify new genes present exclusively in the genomes of these microorganisms. the results obtained from such analyses represent the first genome-wide evaluation regarding genome structure and composition from cvc-associated bacteria, providing additional information about the characteristics of the south american xf gene pool and its relationship with what has been found in north american strains.  genomic comparisons among xf-cvc strains through microarray hybridization analysis microarray hybridization has been widely used to undertake genomic comparisons involving a great number of microorganisms and previous work from our group has established precise criteria to employ this methodology to the study of xf strains, with the aid of an xf 9a5c biochip. thus, we performed similar microarray-based comparisons with different xf strains obtained from citrus plants. four of these xf strains were obtained from cvc-affected trees and are representatives of the most prevalent xf haplotypes found in sweet orange orchards across the state of são paulo, while xf strain cv was obtained from a non-symptomatic tree from the same region. xf strain fb on the other hand, was obtained from a sweet orange tree that displayed symptoms of "pecosita", a disease similar to cvc that occurs in some citrus-growing regions of argentina. the genomic profiles obtained for these strains are depicted in figure which displays a linear representation of the xf strain 9a5c chromosome, followed by orfs present in pxf the large kb chromosome present in xf strain 9a5c. surprisingly, these results showed that, contrary to what had been observed during our former comparisons, most genomic differences within the citrus strains are not associated with deleted orfs, but with elements that are present in greater copy number in the tested strains, when compared to xf strain 9a5c. nonetheless, we were able to confirm the same distribution pattern observed before, since most duplicated orfs are not scattered throughout the genome, but grouped within mobile genetic elements, such as prophages, genomic islands and putative genomic islands previously observed in the genome of xf strain 9a5c. details regarding differences in orf composition, detected by the microarray hybridizations with all xf strains analyzed in this study, can be found in additional file as expected, each strain presents a unique genomic profile, which can be used to characterize all individual strains with high fidelity, as inferred from the high bootstrap values obtained from the cluster analysis shown in figure interestingly, the argentine strain, fb seems to be extremely similar to xf strain 9a5c, as the genomic differences detected between these two strains are restricted to a small number of deleted orfs, distributed along the main chromosome and plasmid pxf all other strains display significantly different profiles, characterized not only by deletions, but also by duplicated orfs. a strong similarity is observed in the profiles obtained for xf strains 56a and c, characterized by scattered duplication of orfs from gi and by what seems to be complete duplications of gi and gi several orfs from prophage xfp and gi are also missing in these two strains. xf strain cv presents a genomic profile that closely resembles xf strains 56a and c, except for the lack of duplications involving orfs within gi xf strain 36f, on the other hand, displays another type of profile, in which no deletions have been detected along the structures of xfp and gi and the duplicated orfs scattered across gi and gi seem to indicate that duplication of these elements is not complete. finally, xf strain 187b displays the most divergent genomic profile, characterized by a very large number of duplicated orfs and mobile elements. once again, duplications seem to span practically the entire structure of gi gi and even gi moreover, several other duplicated regions seem to occur throughout the genome of xf strain 187b, pointing to the possible existence of other mobile genetic elements in xf. interestingly, at least three of these regions seem to map within elements that had been previously identified as putative genomic islands. the two largest xf gis, which were found to be deleted in north american strains are present in all citrus strains analyzed herein and gi seems to be completely duplicated in at least four of the tested xf strains, as well as in two previously analyzed citrus xf strains, x1-b and sj, indicating that this element seems to display intense transpositional activity among representatives of the xf group. interestingly, gi seems to be specific to strains obtained from citrus and coffee trees from south america and may represent a sinapomorphy for the south american xf strains. gi which is also duplicated in other xf strains, displays a region that is similar to the vape-containing region of gi resembling the situation observed with the vap elements from the pathogenic bacterium dichelobacter nodosus. identification of new orfs in the xf-cvc gene pool of citrus-associated xf strains through suppressive subtraction hybridization since the xf biochip used in the experiments described above was based on the genome of xf strain 9a5c, the hybridization experiments can only provide information regarding genes that are present in this strain. thus, to gather information concerning additional orfs present in the gene pool of citrus-associated strains, we employed suppressive subtraction hybridization, using dna from xf strain 9a5c as a driver, against dna from all strains, as described in methods. thus, a total of ssh libraries have been constructed and approximately clones have been obtained and sequenced from these libraries. for each of the strains, the sequenced clones have been trimmed, in order to exclude vector sequences and poor quality regions, and aligned with the aid of cap generating contigs, which contained sequences overall. these contigs span kb of xf sequences, which is equivalent to approximately % of the genome of xf strain 9a5c. we chose to conduct further analyses only with contigs composed by at least different individual sequences and showed size variation from to bp. thus, a total of sequenced clones were excluded from further studies, since they either remained as singlets or grouped into small contigs, composed by a limited number of reads, resulting in poor quality consensus sequences. next, the contigs were filtered against the genome of xf strain 9a5c with the aid of the software cross_match, allowing the identification of stretches of dna that were exclusive to the tested strains. sequencing of approximately ssh clones has allowed the identification of kb of dna sequences that are not present in the genome of xf strain 9a5c. the rate at which novel sequences were identified, as a function of sequenced ssh clones, has been evaluated as described in methods, and judging by the inclination of the curve shown in figure a significant proportion of the novel sequences present in the analyzed genomes is likely to have been identified in this study. these newly identified sequences were analyzed with genemark, to search for individual elements present in their structure, allowing the identification of new orfs, scattered throughout the genomes of the strains analyzed herein. this represents an increment of approximately % in the number of orfs currently known to belong to the gene pool of citrus-associated xf. however, comparative analyses among these newly identified orfs has shown that a large proportion of these elements seemed to belong to different groups of orthologous proteins, present in the genomes of two or more tested strains. thus, to reduce the redundancy of this dataset, the predicted protein sequences from these orfs have been submitted to cluster analysis, as described in methods, resulting in groups of orthologous elements, which are more likely to represent the actual number of new functional genes identified in the gene pool of the tested xf strains. analyses of similarity against the genbank have then been performed with blastx, using the consensus sequences from each goe as input, which allowed the assignment of putative functions for each of these newly identified elements. their distribution into the different functional categories originally described by simpson and co-workers is shown in figure. surprisingly, a relatively small proportion of such sequences has been identified as "no hit". another large fraction of elements has been identified as conserved hypothetical proteins, displaying high similarity to orfs of unknown function, previously identified in the genomes of other microorganisms – particularly in other xf strains. putative functions could be attributed to newly identified goes and the majority of them are directly associated with mobile genetic elements, since we identified new phage structural proteins, recombinases/integrases, elements involved with plasmid replication/stabilization and elements that are homologous to conjugation factors belonging to the traa/trab/cag/vir families, originally described in agrobacterium tumafasciens. the remaining goes, encode proteins that are involved with several metabolic processes of the cell, including a group of six new potential virulence factors, which had not been originally identified in the genome of xf strain 9a5c. these include two homologues for the transcription factor abrb, a new pilin gene, two lpx acetyltransferases and a gene encoding the zonula ocludens toxin from vibrio cholerae. the blastx analyses also confirmed that the sequences from all elements described above could not be found in the genome of xf strain 9a5c. surprisingly, however, these analyses showed that a large proportion of these sequences has already been identified in xf strains from north america – particularly in the case of xf ann- isolated from oleander. as shown in additional file the consensus sequences from goes, showed high similarity to orfs originally described in xf strain ann- encoding both hypothetical proteins and proteins with assigned functions. sixteen goes showed high similarity with elements identified in the genome of xf strain dixon and eight were most similar to sequences found in xf strain temecula-. such an overlap with genes from the north american xf gene pool was unexpected and suggests that the list of unique orfs, recently proposed by doddapaneni and coworkers, as potential targets for strain-specific detection of north american xf strains in the wild must be revised. as shown in table approximately % of the orfs, previously believed to be unique to xf strain ann- and % of the orfs, previously believed to be unique to xf strain dixon have also been found in the citrus-associated strains analyzed in this study. on the other hand, since the consensus sequences from goes returned "no hit" when compared to the ncbi databases, these elements might prove useful as targets for pcr-based detection of citrus-associated xf strains. a as described by doddapaneni and coworkers. altogether, the consensus sequences from only goes showed similarity to genes identified in microorganisms that do not belong to the xf group. interestingly, one third of these elements seem to be homologous to orfs identified in the genome of verminephrobacter eiseniae, a soil-inhabiting bacterium that infects nematodes, while the remaining ones show high similarity to genes found in a great number of microorganisms, such as e. coli, geobacter metalireducens, salmonella enterica, solibacter usitatus, xanthomonas sp., burkholderia sp., chlorobium tepidum, among others. discussion results obtained from several lines of research, developed during the past decade, have turned the phytobacterium xylella fastidiosa into a unique model of study in the fields of both phytopathology and genomics. first of all, this is due to the fact that several diseases, associated with many distinct bacterial strains have been characterized and some of these diseases are responsible for significant economic losses. moreover, a significant amount of genomic information has been obtained for four different strains of this microorganism, allowing the development of both functional and comparative genomic analyses within the group. comparisons performed with the four sequenced xf genomes led doddapaneni and coworkers to suggest that the tenecula- strain genome is the one that most likely resembles the ancestral xf genome, since it has undergone the fewest genetic changes among the four analyzed strains. thus, xf strain 9a5c may have evolved from an ancestral bacterium, carrying a temecula-1-like genome, through incorporation of a series of horizontally transferred elements, such as prophages, gis and plasmids. this possibility is reinforced by the microarray-based comparisons undertaken in this study with the citrus-associated strains, since they provide further evidence for intense transpositional activity of mobile elements during the evolution of this subgroup of xf. so far, most comparative studies among south american xf strains have essentially involved brazilian strains from both citrus and coffee trees. in general, the available data supports the idea that cvc-associated bacteria found in brazil have evolved directly from xf strains that cause coffee leaf scorch, since cvc-associated xf strains have been shown to induce cls symptoms when experimentally inoculated into coffee trees. moreover, it is widely known that most areas in which citrus orchards are presently cultivated used to be dedicated to coffee plantations and there have been reports of cvc vectors feeding on coffee trees. nonetheless, the exact origin of cvc-related xf strains is still a matter of speculation, since very little research has been performed on pecosita-related xf strains. the origin of this subgroup of xf is an important piece of information regarding the evolutionary history of citrus-associated xf strains, specially considering that pecosita has been known to occur in argentina before the first descriptions of cvc in brazil. thus, the present study shows the first genomic-scale comparative evaluation involving a pecosita-related xf strain. it is interesting to verify from these data that, while xf strain fb appears to be very similar to xf strain 9a5c, significant genomic differences have been observed when these strains are compared to xf-cvc bacteria isolated in more recent years, specially regarding the activity of horizontally transferred elements. although it is tempting to speculate from this data that the evolutionary rates among cvc- and pecosita-related strains might differ, it is clear that further phylogenetic and biogeographical studies have yet to be performed in order to shed more light into our knowledge regarding the evolutionary history of the south american xf strains, as well as their corresponding diseases. an attempt to verify the evolutionary relationships between xf strains 9a5c, fb and the other xf strains has been performed by sequencing the 16s-23s rdna spacer region of these bacteria, but since no strain-specific mutations have been found in this sequence, such analysis turned out to be inconclusive. as mentioned before, the fact that the overall genomic information obtained for x. fastidiosa has been mostly based on sequence information derived from north american strains, was likely to have biased to some of the conclusions obtained so far, specially considering the evolutionary complexity of these bacteria, which have been shown to carry an extremely large and active flexible gene pool. as expected, the ssh experiments described in this work, which constitute the first attempt to undertake a large-scale survey of the genomic composition of south american xf strains, showed that a total of orfs, originally identified as unique to the north american strains also seem to be present in the citrus strains from south america. among these orfs, had been proposed to be exclusive to xf strain ann- and were supposed to be present only in the genome of xf strain dixon. thus, sequencing of ssh clones from citrus-associated xf strains resulted in a reduction of approximately 19% in the list of unique genes that could be used as markers for north american strains. these findings reinforce the importance of gathering more information regarding the genomic composition of south american strains – including strains isolated from other hosts, such as coffee, to help in understanding the evolutionary history of the xf group and assist in the development of tools for specific identification of pathovars and other variants. at this point, it is not clear whether the increasing overlap between elements found in south and north american strains are a result of ancestry, or represent lateral transfer events among strains. although the geographical separation of south and north american strains presents a clear barrier to lateral gene transfer events, the evolutionary history of the xf genome has been shown to be extremely dynamic, highly influenced by the activity of transpositional elements from its large flexible gene pool. in fact, direct evidence for the occurrence of genetic exchange between xf strains from the two continents has already been reported after analysis of one xf strain, obtained from plum trees in south america. regardless of its geographical origin, the genomic profile obtained for this strain clearly indicated that it descended from north american strains and was likely to have been accidentally introduced into the south american continent via infected plant material. however, this strain carried practically all genes from pxf a large plasmid found exclusively in south american xf strains, which was likely acquired by conjugation events with south american strains. thus, given the fact that xf strains are known to infect more than plant species – many of which are commercial crops that are constantly shipped to foreign countries, it is not unlikely to assume that other contamination events might have taken place, allowing the introduction of north american strains into the south american territory, and vice versa. further conjugation events, for instance, might have introduced genes that were specific to the north american strains into the gene pool of south american strains, which could have been facilitated by the presence of such a large set of transposable elements in the xf genome and the fact that multiple strains have been shown to coexist in the same host – both in the case of infected plants, as well as insect vectors. the dynamics of xf genome evolution can also be deduced through evaluation of other mobile elements, particularly in the case of gi which seems to be absent from the north american strains, but present in one or more copy numbers in south american strains, as shown in figure among all mobile elements identified in xf, gi is the one that best fulfills the definition of a genomic island, since it displays a higher gc content, altered codon bias, insertion at the 3'end of a trna gene and the presence, at one end, of orfs that display high similarity to a heterodimeric integrase found in association with an insertion element from helicobacter pylori. more interestingly, gi may be significant for the evolution of the xf group, since all studies undertaken so far, involving a total of different south american strains, obtained from both citrus and coffee, indicate that this element may represent a genomic sinapomorphy for the south american xf strains. at this point, it is not possible to determine if elements present in gi play any role in mediating adaptation of south american xf strains to their specific hosts, nor if they participate in the process plant infection and colonization. nonetheless, this element carries a series of orfs whose products are potentially involved with host adaptation and pathogenicity, resembling the structure of pathogenicity islands observed in other bacteria. for instance, gi carries a unique fimbrillin gene, represented by orf xf fimbrillins are components of bacterial type i fimbriae, which are directly implicated in the process of attachment to different types of substrates and biofilm formation. novel fimbriae, composed by distinct fimbrillin genes, are believed to be important virulence determinants, allowing colonization of specific hosts and differentiation of virulent clonal groups of pathogenic bacteria, as in the case of e. coli strains, in which new fimbrillin isomorphs are also associated with horizontally transferred elements. interestingly, xf strains that display specificity to different host and vector species also display a variable number of fimbrilling homologues scattered throughout their genomes. gi has also been shown to carry toxin-associated genes, such as orf xf which encodes a hemolysin-like protein, which is implicated in cytotoxic reactions associated with many pathogenic bacteria, and orf xf involved in the synthesis of the lps fraction of gram-negative bacteria that mediates adverse reactions in both human and animal hosts during infection by pathogenic bacteria. in the case of phytobacteria, the o-antigen portion of lps has been shown to display a strain-specific pattern, which is believed to play a major role in host recognition during the plant colonization process. finally, gi also carries orf xf which encodes the virulence-associated factor vape from dichelobacter nodosus, the causative agent of ovine footrot disease. in this bacterium, the vap genes are present in a family of pathogenicity islands called the vap elements. interestingly, up to different vap elements, varying in length and orf composition, have been mapped throughout the genome of d. nodosus and although their exact function are still unknown, their presence has been clearly shown to be associated with the virulence phenotype in this bacterium. curiously, as mentioned above, the vape-containing region of gi is also duplicated in xf strain 9a5c, as a part of gi resembling the organization of vap elements in d. nodosus. as mentioned above, additional virulence factors have now been identified in the genome of citrus-associated strains through the ssh experiments. one of these elements encodes a new form of pilin, associated with type iv fimbriae, whose twitching motility mechanism has been shown to be of capital importance to host colonization in xf. this finding reinforces the importance of fimbriae for the evolutionary divergence of xf strains. transcriptional regulators, such as the abrb activator have also been found through the ssh experiments. this transcription factor is responsible for controlling several genes, specifically activated at the end of bacterial exponential growth phase in bacillus subtilis, and has been shown to be implicated with biofilm formation in this bacterium. interestingly, biofilm formation is believed to be an important virulence factor in xf during the development of both cvc and pd, since growth in biofilm is likely to participate in the process of bacterial adherence to the xylem vessels, contribute to xylem occlusion and increase bacterial survival against the oxidative burst mediated by infected plant tissues. two different homologues of the abrb gene have been found in out of the analyzed xf strains, while the new pilin gene has been found only in xf strain c. two other potential virulence factors found in the xf-cvc gene pool seem to belong to the cyse/laca/lpxa/nodl family of acetyltransferases, which are characterized by multiple repeats of the sequence -g-x. in both cases, the highest similarities found for these newly identified acetyltransferases are with genes lpxa and lpxd from the north american strains ann- and temecula-, which are involved in synthesis and modification of the o-antigen fraction of gram-negative bacteria lps. as mentioned above, the strain-specific structure of the o-antigen has been show to be an important mediator in plant colonization by symbiotic bacteria, especially in the case of nodule formation during rhizobium-legume interactions. finally, the genomes of xf strains 187b and c seem to carry a copy of the gene that encodes the zonula occludens toxin, also found in xf strain ann- zot-like genes have been found in association with several bacterial pathogens, such as xanthomonas, vibrio and stenotrophomonas, among others and are likely to be laterally transferred among microorganisms through the action of filamentous phages. this toxin has been originally described as an important virulence factor in vibrio cholerae, and seems to be responsible for the development of severe cases of diarrhea caused by v. cholerae strains that do not carry the cholera toxin gene ctxa. the activity of zot as an enterotoxin seems to be associated to its capacity to interfere with tight junctions of the gastrointestinal epithelium, altering its permeability to water and other substances. nonetheless, regardless of its recognized importance as a virulence factor in animal or human infections, there is no direct evidence that zot plays any role during plant colonization and/or pathogenicity, although this gene has also been found in the genome of xanthomonas campestris, the causative agent of black rot disease, characterized by hyper-hydration of infected plant tissues, associated with wet edged lesions, which may be a result of zot activity.  the experiments described in the present study represent the first attempt to conduct microarray-based genomic comparisons and a large-scale survey of genes present in the genomes of south american xf strains. the results from the microarray-based comparison provide further evidence concerning the intense transpositional activity of several horizontally transferred elements and reinforce previous studies regarding the importance of lateral gene transfer as a major mediator in the evolution of this important group of phytopathogens. moreover, comparison of the microarray-based genomic profiles showed similarity between xf strains 9a5c and fb which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. it is clear that a more comprehensive evaluation of both coffee- and pecosita-related strains is necessary before we can fully understand the evolutionary history of south american xf strains and their associated diseases. the characterization of approximately ssh clones, from six representatives of citrus haplotypes, have now provided a more comprehensive view of the size and composition of the xf-cvc gene pool, allowing us to identify new orfs – a number that represents an ~ % increase in our current knowledge of the south american xf gene pool. these results allowed identification of new putative virulence factors, as well as novel potential markers for strain identification within this subgroup of phytopathogens by molecular-based approaches. moreover, several sequences previously believed to be unique markers for north american strains have now been found in the genomes of these south american strains. these new data point to the necessity of revising the molecular markers currently accepted as potential targets for identification of distinct xf strains in the wild. finally, by evaluating the rate at which novel sequences have been identified through the ssh approach, we conclude that the complete composition of the south american xf gene pool can still be stretched, specially if this type of analysis is further extended to strains obtained from alternative hosts, such as coffee, which is known to harbor many different xf strains throughout the south american territory. 
nonetheless, although significant amounts of information have been generated from xf strains, a large proportion of these efforts has concentrated on the study of north american strains, limiting our understanding about the genomic composition of south american strains – which is particularly important for cvc-associated strains. moreover, a large-scale sequencing effort, based on suppressive subtraction hybridization, identified new orfs, distributed in groups of orthologous elements, throughout the genomes of these bacteria. results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of xf. comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to south american strains. the xylem-inhabiting bacterium xylella fastidiosa is the causal agent of pierce's disease in vineyards and citrus variegated chlorosis in orange trees. the newly identified orfs, obtained by ssh, represent an approximately 10% increase in our current knowledge of the south american xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. this sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of xf to infect different hosts, causing a variety of symptoms. this paper describes the first genome-wide comparison among south american xf strains, involving distinct citrus-associated bacteria. surprisingly, this list of novel elements include sequences previously believed to be unique to north american strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct xf strains. moreover, the microarray-based genomic profiles showed similarity between xf strains 9a5c and fb which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with xf strains.
 the xylem-inhabiting bacterium xylella fastidiosa is the causal agent of pierce's disease in vineyards and citrus variegated chlorosis in orange trees. both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with xf strains. this sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of xf to infect different hosts, causing a variety of symptoms. nonetheless, although significant amounts of information have been generated from xf strains, a large proportion of these efforts has concentrated on the study of north american strains, limiting our understanding about the genomic composition of south american strains – which is particularly important for cvc-associated strains.  this paper describes the first genome-wide comparison among south american xf strains, involving distinct citrus-associated bacteria. comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to south american strains. moreover, a large-scale sequencing effort, based on suppressive subtraction hybridization, identified new orfs, distributed in groups of orthologous elements, throughout the genomes of these bacteria.  results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of xf. moreover, the microarray-based genomic profiles showed similarity between xf strains 9a5c and fb which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. the newly identified orfs, obtained by ssh, represent an approximately 10% increase in our current knowledge of the south american xf gene pool and include new putative virulence factors, as well as novel potential markers for strain identification. surprisingly, this list of novel elements include sequences previously believed to be unique to north american strains, pointing to the necessity of revising the list of specific markers that may be used for identification of distinct xf strains. 