BACKGROUND
of the over  <dig> salmonella serotypes recognized to date  <cit> , about  <dig> infect humans and other warm-blooded vertebrates, which are classified into salmonella subgroup i .
although most of these salmonella serotypes cause self-limiting gastroenteritis in humans, four cause typhoid fever, a serious and potentially fatal systemic infection, including s. typhi  and s. paratyphi a, b and c. it is a long unanswered question whether different salmonella typhoid agents cause the disease by similar or distinct mechanisms.
as all salmonella share high levels of genetic similarity, it is possible to reveal genetic differences or similarities in pathogenicity among the salmonella typhoid agents by focusing on genomic features common to some or all of the salmonella typhoid agents.
the extraordinary levels of genetic similarity among salmonellae were recognized first by genomic dna re-association experiments  <cit> , later by physical mapping  <cit> , and recently by whole genome sequencing  <cit> .
in the mid-1990s, physical mapping revealed an overall common genome structure among all salmonella; it also revealed special features of individual salmonella lineages, such as the major genomic insertions and rearrangements of s. typhi  <cit>  and s. paratyphi a  <cit> , which were later all confirmed by whole genome sequencing  <cit> .
sequence comparison of the salmonella genomes further reveals that about 13% of the genes in s. typhi are not found in s. typhimurium  <cit> , and many of these serovar-specific genes were apparently acquired by lateral transfer of large dna segments from other sources  <cit> , which, if  <dig> kb or larger, can be resolved on the physical maps  <cit> .
several lines of evidence suggest that the typhoid agents have not evolved by vertical descent of one from another, as genetic distances are not closer among the typhoid agents than each of them to the non-typhoidal salmonellae  <cit> .
however, they may have common or similar routes of acquisition of genes coding for the typhoid pathogenicity.
comparison of multiple typhoid agents may lead to identification of these genes.
s. paratyphi c is a member of serogroup c <dig>  <cit> , causing typhoid in humans but, unlike s. typhi, also occasionally infecting animals  <cit> .
previously we reported i-ceui maps on two s. paratyphi c strains, rks <dig> and rks <dig>  which are very different in size and organization of the seven i-ceui fragments.
in several ways, s. paratyphi c is similar to s. typhi, including the possession of a large pathogenicity island, spi <dig>  with genes coding for the vi  antigen and other genes potentially associated with virulence  <cit> .
it is thus of great significance to reveal the genomic features of s. paratyphi c at higher resolution and involving a broader range of wild type strains, and then compare them with those of s. typhi to explore the evolution of the typhoid-associated pathogenicity.
in this paper, we first report a high resolution genome map of a representative s. paratyphi c strain, rks <dig>  featured with several insertions, deletions and rearrangements.
we then present genome maps of additional wild type strains of s. paratyphi c constructed with i-ceui, which reveal diverse genome structures among the wild type strains of s. paratyphi c. some strains have deletions in the highly conserved mete-arge region, which have not previously been observed in any other salmonella strains.
RESULTS
cleavage of s. paratyphi c rks <dig> genomic dna with i-ceui
i-ceui is an intron-encoded endonuclease, with cleavage sites in rrl genes  <cit> .
cleavage of s. paratyphi c rks <dig> genomic dna with i-ceui generated seven fragments .
this number is consistent with the previously analyzed salmonella genomes  <cit> , as all salmonella lineages have seven rrl genes.
in figure 1a, there are bright as well as less bright dna bands, the latter being labeled as combinations of two or three of the seven bright bands.
these less bright dna bands are partial cleavage products showing the neighboring relationships of the seven i-ceui cleaved dna segments.
analyses of both complete and partial i-ceui cleavages resulted in the cleavage map of s. paratyphi c rks <dig> in figure 1b.
in designation of the i-ceui fragments, the same letters were used to label homologous dna segments in different salmonella lineages.
in s. typhimurium lt <dig>  the order of the seven i-ceui fragments is abcdefg clockwise, but in s. paratyphi c rks <dig> the order is abcfdeg due to the translocation of fragment f. translocations of i-ceui fragments are usually mediated by homologous recombination between pairs of the rrn genes  <cit> , which usually have greater than 99% sequence identity.
the translocation of i-ceui f in s. paratyphi c rks <dig> led to three hybrid rrn operons.
the rrl genes are used to exemplify the hybrid rrn operons in figure 1b, shown as rrlc/b, rrle/c and rrlb/e, but recombination may also occur in rrs.
xbai and avrii cleavages and transposon tn <dig> insertion analysis of the genome
xbai cleavage generated  <dig> fragments, ranging from  <dig> kb  to  <dig> kb .
avrii cleavage generated  <dig> fragments, ranging from  <dig> kb  to  <dig> kb .
to determine the order of fragments in both xbai and avrii cleavages and locate genes on the genome of s. paratyphi c, we transferred the transposon tn <dig> that was inserted in known genes in s. typhimurium lt <dig>  <cit>  to the s. paratyphi c background by bacteriophage p <dig> mediated transduction as previously described  <cit> , but with the introduction of o <dig> antigen-encoding genes to make s. paratyphi c sensitive to p <dig> .
since the tn <dig> sequence contains both xbai and avrii cleavage sites  <cit> , the location of a tn <dig> insertion can be determined by xbai or avrii cleavage and pfge analyses.
dna from a strain with a tn <dig> insertion in a specific fragment will lose that fragment, and two fragments will be detected that sum to the size of the lost fragment plus  <dig>  kb, which is the size of tn <dig>  we determined locations for  <dig> genes, as shown in figure  <dig>
a high resolution genome map of s. paratyphi c rks4594
after summarizing experimental results obtained by cleavages with i-ceui, xbai and avrii, tn <dig> insertion analyses and double digestions , we constructed a high resolution genome map for s. paratyphi c rks <dig> .
xbai fragment p  was not mapped on the chromosome; it is a plasmid  with a copy number of  <dig> to  <dig> as judged by brightness of the dna band when compared with other bands on a pfge gel.
in addition to an xbai cleavage site, this plasmid also has an avrii site .
most genes are in the same order as in s. typhimurium lt <dig>  with the following exceptions.
the translocation of i-ceui fragment f moves the thic gene  to a new location; a very large inversion  includes purg  and pepn .
on the genome of s. typhimurium lt <dig>  the region clockwise to pncb  is occupied by prophage gifsy- <dig> , and the region counterclockwise to nadb  is occupied by prophage gifsy- <dig> .
the distance between pncb and purg  plus that between pepn and nadb  in s. paratyphi c rks <dig>  roughly equals the distance between pncb and pepn  plus that between purg and nadb  in s. typhimurium lt <dig> .
so, judged by the distances between genes, it seems that two prophages with sizes similar to those of gifsy- <dig> and gifsy- <dig> are present in s. paratyphi c rks <dig>  although their identities are unknown.
because the inversion seems to include most genes of the two prophages, and because the prophages are inverted in orientation in s. typhimurium lt <dig>  we speculate that the exchange points of the inversion were between genes in the two prophages.
at the position of gifsy- <dig> in s. typhimurium lt <dig>  s. typhi ct <dig> has a different phage sequence, but at the position of gifsy- <dig> in s. typhimurium lt <dig>  s. typhi ct <dig> does not have a phage sequence  <cit> .
a similar inversion has not been observed in other salmonella serovars.
there are several genomic regions with increased distances between genes relative to s. typhimurium lt <dig>  which we speculate to be dna segments acquired from foreign sources during the evolution of s. paratyphi c. the first  is  <dig> kb between purg and guaa, and the second  is  <dig> kb between purc and purf.
it is possible that both are prophages as judged by their sizes, or that one or both might be summations of smaller insertions in those regions from different sources.
a  <dig> kb genomic increase  was located in the region between chea and dadx.
in this region, there is a  <dig> kb phage-associated dna segment in s. typhi ct <dig> but not in s. typhimurium lt <dig>  <cit> .
it is not known whether the  <dig> kb is part of the  <dig> kb phage dna or whether the whole  <dig> kb phage dna is here and there is a  <dig> kb deletion, collectively giving rise to a net  <dig> kb increase of dna in that region.
the largest insertion is the one between melr and mutl, about  <dig> kb, in a location similar to that of spi <dig> in s. typhi  <cit> .
as it is previously known that the genome of s. paratyphi c carries a shorter version of spi <dig>  <cit> , we assume that this  <dig> kb insertion probably is the s. paratyphi c version of spi <dig>
we also located genomic regions of reduced distances  between genes in s. paratyphi c rks <dig> when compared with s. typhimurium lt <dig>  starting from the gene thr and moving clockwise, the first possible deletion is one about  <dig> kb between ahpc and nada and the second one is about  <dig> kb between bioa and aroa.
whether they contain single deletions or several smaller deletions would both be interesting, especially regarding the gene loss involved, as the absence of some of the genes may be associated with host restriction.
the distance between arod and pyrf is  <dig> kb in s. typhimurium lt <dig> but only  <dig> kb in s. paratyphi c rks <dig>  in the same region on the genome of s. typhi ct <dig>  there is a  <dig> kb phage-associated dna sequence.
depending on whether s. paratyphi c rks <dig> contains this  <dig> kb phage dna, this organism has a deletion of  <dig> kb or  <dig> kb in this region relative to s. typhimurium lt <dig>
s. typhimurium lt <dig> has prophage fels- <dig>  between genes tyra and tcte.
in this region, s. paratyphi c rks <dig> is  <dig> kb shorter compared to s. typhimurium lt <dig>  if fels- <dig> is not present, there should be another  <dig> kb deletion in s. paratyphi c rks <dig>  three additional regions of possible deletions of  <dig> kb ,  <dig> kb  and  <dig> kb  need further characterization.
although we do not completely rule out the possibility that the reduced distances between genes actually resulted from translocation of dna segments instead of deletions, it is very unlikely, as gene order is highly conserved in salmonella and dna translocation does not generally occur except those mediated by recombination of rrn genes or other repeated elements.
plastic genome structure of s. paratyphi c
to determine whether the genome structure of s. paratyphi c is uniform and stable among the populations like that of s. paratyphi a  <cit> , or is diverse and plastic like that of s. typhi  <cit> , we made i-ceui maps on s. paratyphi c isolates gathered from different geographical areas over a broad range of time.
as shown in figure 4a, individual s. paratyphi c strains have obviously different pfge patterns, especially regarding the partial cleavage bands, which reflect different orders of the genomic dna segments .
rather strikingly, two strains, rks <dig> and rks <dig>  have a  <dig> kb deletion in a region between rrla and rrlb.
this region is extremely stable in salmonella and no deletion has been reported in other salmonella lineages.
currently, we do not know what genes have been deleted in these two strains and whether they have identical deletions.
discussion
the existence of different typhoid agents causing very similar clinical manifestations presents exceptional opportunities for elucidating their origin and for understanding the molecular mechanisms of host adaptability.
two of the four human typhoid agents, s. typhi and s. paratyphi a, have been studied at the level of physical genome maps with a focus on genome structure in large numbers of isolates  <cit>  and at the level of whole genome sequence with a focus on individual genes and their regulation in representative strains  <cit> .
when compared with s. typhimurium, s. typhi has over  <dig> unique genes  <cit> .
it is reasonable to speculate that some of the s. typhi-specific genes may be directly involved in typhoid pathogenicity, although cooperation with genes of the "core salmonella genome", i.e., genes shared by most salmonella lineages, should also be important.
if one assumes that the typhoid agents share some or many typhoid-associated genes, then comparisons among them should help in identification.
comparative studies between s. typhi and s. paratyphi a, however, are so far not conclusive, because they each have many genomic features not shared by the other.
therefore, it is necessary to compare more than two salmonella typhoidal lineages to further highlight some possible common genomic features.
additionally, it is also possible that some typhoid agents may have evolved vertically from a common ancestor and others may have acquired typhoid pathogenicity horizontally.
systematic genome comparisons among multiple typhoid agents are rational approaches toward elucidating these evolutionary processes.
the main purpose of this study was to reveal large scale genomic features of a representative s. paratyphi c strain and overall genome structure of s. paratyphi c populations.
on the high resolution physical map of s. paratyphi c rks <dig>  we resolved four possible genomic insertions, totaling  <dig> kb, and seven possible deletions, totaling  <dig> kb, relative to s. typhimurium lt <dig>  at this stage, we do not know what genes are contained in the insertions and what genes are involved and deletions, but localization of these insertions and deletions on the genome will significantly facilitate comparative studies among the typhoid agents.
a basic feature of the genome structure of s. paratyphi c is its plasticity – large genomic dna segments are arranged differently in different wild type strains.
in strain rks <dig>  we first revealed two rearrangement events, including inversion of a  <dig> kb segment covering the ter region and the translocation of the  <dig> kb i-ceui f fragment from its location between i-ceui e and g as in s. typhimurium and most other salmonella genomes to the location between i-ceui c and d, leading to the order i-ceui abcfdeg in s. paratyphi c .
diverse genome structures among the s. paratyphi c wild type strains representing different populations have resulted from homologous recombination between rrn genes in all analyzed strains except rks <dig>  in which a very rare inversion occurred between a site in i-ceui fragment b and a site in i-ceui fragment c; we are now determining the exact locations and identities of the exchange end points .
the previously analyzed typhoid agents have different situations regarding the stability of genome structure.
we first reported the rearranged genome structure of s. typhi ty <dig>  <cit>  and then found that wild type strains of s. typhi representing different populations have diverse genome structures; we called this phenomenon "genomic plasticity"  <cit> .
our interpretation of this phenomenon is that large insertions disrupt the physical balance of the genome between ori and ter, and rearrangements will rebalance the genome  <cit> .
in contrast, the other extensively investigated typhoid agent, s. paratyphi a, at both physical map and sequence levels, has a uniform genome structure among different isolates, even though this organism does have insertions totaling  <dig> kb clustered together on the genome.
interestingly, the  <dig> kb inversion, seen in all examined wild type strains of s. paratyphi a, nearly completely restored the genomic balance  <cit> , making it unnecessary for the bacteria to further rearrange the genome.
the situation with s. paratyphi b, on the other hand, seems much more complex, because, unlike s. typhi, s. paratyphi a or s. paratyphi c, which are all monophyletic  <cit> , bacteria under the name s. paratyphi b seem to be genetically diverse.
for example, the d-tartrate-positive subline  causes gastroenteritis instead of typhoid.
kauffmann proposed the name s. java for this subline  <cit> , however le minor et al.
proposed that the name s. java be dropped  <cit> .
even within the d-tartrate-negative and typhoid-causing subline, the diversity in genome structure, as reflected by xbai and avrii cleavage patterns, is also significant  <cit> .
genome sequencing of a representative typhoid-causing strain of s. paratyphi b followed by population analysis will provide insights into the phylogenetics and evolution of s. paratyphi b as a typhoid agent.
this study demonstrates that s. paratyphi c is similar to s. typhi in the plasticity of genome structure.
however, such a similarity is not indicative of their relatedness.
instead, this similarity suggests a similar route of evolution: acquisition of typhoid causing genes, disruption of physical balance of the genome, and rearrangement of the genome to restore the balance.
this route may apply to s. paratyphi a as well: the non-plastic feature of its genome may be merely a result of the near complete restoration of the genome balance by the  <dig> kb inversion.
it is not known at present whether s. paratyphi b, monophyletic or polyphyletic as it may turn out to be, has a stable or plastic genome structure; however, it may too have followed a similar route of pathogenic evolution.
revelation of genes that contribute to the typhoid pathogenesis in the individual typhoid agents will update our knowledge on pathogenically similar but phylogenetically not directly related bacteria.
for achieving this goal, sequencing of additional salmonella typhoid agents is currently underway in several institutions, and some of the sequencing projects are close to or already at the finishing stages.
this study also revealed an unusual feature of s. paratyphi c: unlike s. typhi wild type strains that do not have significant dna content differences except spi <dig> that is missing from some s. typhi isolates  <cit> , two s. paratyphi c strains in this study have  <dig> kb deletions in a normally extremely stable genomic region between rrla and rrlb .
this finding suggests that genes in the deleted  <dig> kb dna are no longer needed at least in some s. paratyphi c strains, or they are not needed by any of the typhoid agents in general.
it is possible that, considering the short evolutionary history of the salmonella typhoid agents  <cit> , these bacteria may have not yet eliminated all of the non-selected genes, although some stochastic factors, such as integration or excision of prophages that make genomic rebalancing necessary, may have worked to help streamline the genome by removing some of the presumably unneeded genes before they even had a chance to become pseudogenes.
these findings may help prioritize genes for functional analyses by excluding large numbers of genes from further study.
CONCLUSIONS
s. paratyphi c has diverse genome structures, possibly as a result of large genomic insertions, e.g., spi <dig>  although the salmonella typhoid agents may not be more closely related among them than each of them to other salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes and making genome structure rearrangements.
comparison of multiple salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes.

 of the over salmonella serotypes recognized to date, about infect humans and other warm-blooded vertebrates, which are classified into salmonella subgroup i. although most of these salmonella serotypes cause self-limiting gastroenteritis in humans, four cause typhoid fever, a serious and potentially fatal systemic infection, including s. typhi and s. paratyphi a, b and c. it is a long unanswered question whether different salmonella typhoid agents cause the disease by similar or distinct mechanisms. as all salmonella share high levels of genetic similarity, it is possible to reveal genetic differences or similarities in pathogenicity among the salmonella typhoid agents by focusing on genomic features common to some or all of the salmonella typhoid agents. the extraordinary levels of genetic similarity among salmonellae were recognized first by genomic dna re-association experiments, later by physical mapping, and recently by whole genome sequencing. in the mid-1990s, physical mapping revealed an overall common genome structure among all salmonella; it also revealed special features of individual salmonella lineages, such as the major genomic insertions and rearrangements of s. typhi and s. paratyphi a, which were later all confirmed by whole genome sequencing. sequence comparison of the salmonella genomes further reveals that about 13% of the genes in s. typhi are not found in s. typhimurium, and many of these serovar-specific genes were apparently acquired by lateral transfer of large dna segments from other sources, which, if kb or larger, can be resolved on the physical maps. several lines of evidence suggest that the typhoid agents have not evolved by vertical descent of one from another, as genetic distances are not closer among the typhoid agents than each of them to the non-typhoidal salmonellae. however, they may have common or similar routes of acquisition of genes coding for the typhoid pathogenicity. comparison of multiple typhoid agents may lead to identification of these genes. s. paratyphi c is a member of serogroup c, causing typhoid in humans but, unlike s. typhi, also occasionally infecting animals. previously we reported i-ceui maps on two s. paratyphi c strains, rks and rks which are very different in size and organization of the seven i-ceui fragments. in several ways, s. paratyphi c is similar to s. typhi, including the possession of a large pathogenicity island, spi with genes coding for the vi antigen and other genes potentially associated with virulence. it is thus of great significance to reveal the genomic features of s. paratyphi c at higher resolution and involving a broader range of wild type strains, and then compare them with those of s. typhi to explore the evolution of the typhoid-associated pathogenicity. in this paper, we first report a high resolution genome map of a representative s. paratyphi c strain, rks featured with several insertions, deletions and rearrangements. we then present genome maps of additional wild type strains of s. paratyphi c constructed with i-ceui, which reveal diverse genome structures among the wild type strains of s. paratyphi c. some strains have deletions in the highly conserved mete-arge region, which have not previously been observed in any other salmonella strains.  cleavage of s. paratyphi c rks genomic dna with i-ceui i-ceui is an intron-encoded endonuclease, with cleavage sites in rrl genes. cleavage of s. paratyphi c rks genomic dna with i-ceui generated seven fragments. this number is consistent with the previously analyzed salmonella genomes, as all salmonella lineages have seven rrl genes. in figure 1a, there are bright as well as less bright dna bands, the latter being labeled as combinations of two or three of the seven bright bands. these less bright dna bands are partial cleavage products showing the neighboring relationships of the seven i-ceui cleaved dna segments. analyses of both complete and partial i-ceui cleavages resulted in the cleavage map of s. paratyphi c rks in figure 1b. in designation of the i-ceui fragments, the same letters were used to label homologous dna segments in different salmonella lineages. in s. typhimurium lt the order of the seven i-ceui fragments is abcdefg clockwise, but in s. paratyphi c rks the order is abcfdeg due to the translocation of fragment f. translocations of i-ceui fragments are usually mediated by homologous recombination between pairs of the rrn genes, which usually have greater than 99% sequence identity. the translocation of i-ceui f in s. paratyphi c rks led to three hybrid rrn operons. the rrl genes are used to exemplify the hybrid rrn operons in figure 1b, shown as rrlc/b, rrle/c and rrlb/e, but recombination may also occur in rrs. xbai and avrii cleavages and transposon tn insertion analysis of the genome xbai cleavage generated fragments, ranging from kb to kb. avrii cleavage generated fragments, ranging from kb to kb. to determine the order of fragments in both xbai and avrii cleavages and locate genes on the genome of s. paratyphi c, we transferred the transposon tn that was inserted in known genes in s. typhimurium lt to the s. paratyphi c background by bacteriophage p mediated transduction as previously described, but with the introduction of o antigen-encoding genes to make s. paratyphi c sensitive to p. since the tn sequence contains both xbai and avrii cleavage sites, the location of a tn insertion can be determined by xbai or avrii cleavage and pfge analyses. dna from a strain with a tn insertion in a specific fragment will lose that fragment, and two fragments will be detected that sum to the size of the lost fragment plus kb, which is the size of tn we determined locations for genes, as shown in figure a high resolution genome map of s. paratyphi c rks4594 after summarizing experimental results obtained by cleavages with i-ceui, xbai and avrii, tn insertion analyses and double digestions, we constructed a high resolution genome map for s. paratyphi c rks. xbai fragment p was not mapped on the chromosome; it is a plasmid with a copy number of to as judged by brightness of the dna band when compared with other bands on a pfge gel. in addition to an xbai cleavage site, this plasmid also has an avrii site. most genes are in the same order as in s. typhimurium lt with the following exceptions. the translocation of i-ceui fragment f moves the thic gene to a new location; a very large inversion includes purg and pepn. on the genome of s. typhimurium lt the region clockwise to pncb is occupied by prophage gifsy-, and the region counterclockwise to nadb is occupied by prophage gifsy-. the distance between pncb and purg plus that between pepn and nadb in s. paratyphi c rks roughly equals the distance between pncb and pepn plus that between purg and nadb in s. typhimurium lt. so, judged by the distances between genes, it seems that two prophages with sizes similar to those of gifsy- and gifsy- are present in s. paratyphi c rks although their identities are unknown. because the inversion seems to include most genes of the two prophages, and because the prophages are inverted in orientation in s. typhimurium lt we speculate that the exchange points of the inversion were between genes in the two prophages. at the position of gifsy- in s. typhimurium lt s. typhi ct has a different phage sequence, but at the position of gifsy- in s. typhimurium lt s. typhi ct does not have a phage sequence. a similar inversion has not been observed in other salmonella serovars. there are several genomic regions with increased distances between genes relative to s. typhimurium lt which we speculate to be dna segments acquired from foreign sources during the evolution of s. paratyphi c. the first is kb between purg and guaa, and the second is kb between purc and purf. it is possible that both are prophages as judged by their sizes, or that one or both might be summations of smaller insertions in those regions from different sources. a kb genomic increase was located in the region between chea and dadx. in this region, there is a kb phage-associated dna segment in s. typhi ct but not in s. typhimurium lt. it is not known whether the kb is part of the kb phage dna or whether the whole kb phage dna is here and there is a kb deletion, collectively giving rise to a net kb increase of dna in that region. the largest insertion is the one between melr and mutl, about kb, in a location similar to that of spi in s. typhi. as it is previously known that the genome of s. paratyphi c carries a shorter version of spi, we assume that this kb insertion probably is the s. paratyphi c version of spi we also located genomic regions of reduced distances between genes in s. paratyphi c rks when compared with s. typhimurium lt starting from the gene thr and moving clockwise, the first possible deletion is one about kb between ahpc and nada and the second one is about kb between bioa and aroa. whether they contain single deletions or several smaller deletions would both be interesting, especially regarding the gene loss involved, as the absence of some of the genes may be associated with host restriction. the distance between arod and pyrf is kb in s. typhimurium lt but only kb in s. paratyphi c rks in the same region on the genome of s. typhi ct there is a kb phage-associated dna sequence. depending on whether s. paratyphi c rks contains this kb phage dna, this organism has a deletion of kb or kb in this region relative to s. typhimurium lt s. typhimurium lt has prophage fels- between genes tyra and tcte. in this region, s. paratyphi c rks is kb shorter compared to s. typhimurium lt if fels- is not present, there should be another kb deletion in s. paratyphi c rks three additional regions of possible deletions of kb, kb and kb need further characterization. although we do not completely rule out the possibility that the reduced distances between genes actually resulted from translocation of dna segments instead of deletions, it is very unlikely, as gene order is highly conserved in salmonella and dna translocation does not generally occur except those mediated by recombination of rrn genes or other repeated elements. plastic genome structure of s. paratyphi c to determine whether the genome structure of s. paratyphi c is uniform and stable among the populations like that of s. paratyphi a, or is diverse and plastic like that of s. typhi, we made i-ceui maps on s. paratyphi c isolates gathered from different geographical areas over a broad range of time. as shown in figure 4a, individual s. paratyphi c strains have obviously different pfge patterns, especially regarding the partial cleavage bands, which reflect different orders of the genomic dna segments. rather strikingly, two strains, rks and rks have a kb deletion in a region between rrla and rrlb. this region is extremely stable in salmonella and no deletion has been reported in other salmonella lineages. currently, we do not know what genes have been deleted in these two strains and whether they have identical deletions. discussion the existence of different typhoid agents causing very similar clinical manifestations presents exceptional opportunities for elucidating their origin and for understanding the molecular mechanisms of host adaptability. two of the four human typhoid agents, s. typhi and s. paratyphi a, have been studied at the level of physical genome maps with a focus on genome structure in large numbers of isolates and at the level of whole genome sequence with a focus on individual genes and their regulation in representative strains. when compared with s. typhimurium, s. typhi has over unique genes. it is reasonable to speculate that some of the s. typhi-specific genes may be directly involved in typhoid pathogenicity, although cooperation with genes of the "core salmonella genome", i.e., genes shared by most salmonella lineages, should also be important. if one assumes that the typhoid agents share some or many typhoid-associated genes, then comparisons among them should help in identification. comparative studies between s. typhi and s. paratyphi a, however, are so far not conclusive, because they each have many genomic features not shared by the other. therefore, it is necessary to compare more than two salmonella typhoidal lineages to further highlight some possible common genomic features. additionally, it is also possible that some typhoid agents may have evolved vertically from a common ancestor and others may have acquired typhoid pathogenicity horizontally. systematic genome comparisons among multiple typhoid agents are rational approaches toward elucidating these evolutionary processes. the main purpose of this study was to reveal large scale genomic features of a representative s. paratyphi c strain and overall genome structure of s. paratyphi c populations. on the high resolution physical map of s. paratyphi c rks we resolved four possible genomic insertions, totaling kb, and seven possible deletions, totaling kb, relative to s. typhimurium lt at this stage, we do not know what genes are contained in the insertions and what genes are involved and deletions, but localization of these insertions and deletions on the genome will significantly facilitate comparative studies among the typhoid agents. a basic feature of the genome structure of s. paratyphi c is its plasticity – large genomic dna segments are arranged differently in different wild type strains. in strain rks we first revealed two rearrangement events, including inversion of a kb segment covering the ter region and the translocation of the kb i-ceui f fragment from its location between i-ceui e and g as in s. typhimurium and most other salmonella genomes to the location between i-ceui c and d, leading to the order i-ceui abcfdeg in s. paratyphi c. diverse genome structures among the s. paratyphi c wild type strains representing different populations have resulted from homologous recombination between rrn genes in all analyzed strains except rks in which a very rare inversion occurred between a site in i-ceui fragment b and a site in i-ceui fragment c; we are now determining the exact locations and identities of the exchange end points. the previously analyzed typhoid agents have different situations regarding the stability of genome structure. we first reported the rearranged genome structure of s. typhi ty and then found that wild type strains of s. typhi representing different populations have diverse genome structures; we called this phenomenon "genomic plasticity". our interpretation of this phenomenon is that large insertions disrupt the physical balance of the genome between ori and ter, and rearrangements will rebalance the genome. in contrast, the other extensively investigated typhoid agent, s. paratyphi a, at both physical map and sequence levels, has a uniform genome structure among different isolates, even though this organism does have insertions totaling kb clustered together on the genome. interestingly, the kb inversion, seen in all examined wild type strains of s. paratyphi a, nearly completely restored the genomic balance, making it unnecessary for the bacteria to further rearrange the genome. the situation with s. paratyphi b, on the other hand, seems much more complex, because, unlike s. typhi, s. paratyphi a or s. paratyphi c, which are all monophyletic, bacteria under the name s. paratyphi b seem to be genetically diverse. for example, the d-tartrate-positive subline causes gastroenteritis instead of typhoid. kauffmann proposed the name s. java for this subline, however le minor et al. proposed that the name s. java be dropped. even within the d-tartrate-negative and typhoid-causing subline, the diversity in genome structure, as reflected by xbai and avrii cleavage patterns, is also significant. genome sequencing of a representative typhoid-causing strain of s. paratyphi b followed by population analysis will provide insights into the phylogenetics and evolution of s. paratyphi b as a typhoid agent. this study demonstrates that s. paratyphi c is similar to s. typhi in the plasticity of genome structure. however, such a similarity is not indicative of their relatedness. instead, this similarity suggests a similar route of evolution: acquisition of typhoid causing genes, disruption of physical balance of the genome, and rearrangement of the genome to restore the balance. this route may apply to s. paratyphi a as well: the non-plastic feature of its genome may be merely a result of the near complete restoration of the genome balance by the kb inversion. it is not known at present whether s. paratyphi b, monophyletic or polyphyletic as it may turn out to be, has a stable or plastic genome structure; however, it may too have followed a similar route of pathogenic evolution. revelation of genes that contribute to the typhoid pathogenesis in the individual typhoid agents will update our knowledge on pathogenically similar but phylogenetically not directly related bacteria. for achieving this goal, sequencing of additional salmonella typhoid agents is currently underway in several institutions, and some of the sequencing projects are close to or already at the finishing stages. this study also revealed an unusual feature of s. paratyphi c: unlike s. typhi wild type strains that do not have significant dna content differences except spi that is missing from some s. typhi isolates, two s. paratyphi c strains in this study have kb deletions in a normally extremely stable genomic region between rrla and rrlb. this finding suggests that genes in the deleted kb dna are no longer needed at least in some s. paratyphi c strains, or they are not needed by any of the typhoid agents in general. it is possible that, considering the short evolutionary history of the salmonella typhoid agents, these bacteria may have not yet eliminated all of the non-selected genes, although some stochastic factors, such as integration or excision of prophages that make genomic rebalancing necessary, may have worked to help streamline the genome by removing some of the presumably unneeded genes before they even had a chance to become pseudogenes. these findings may help prioritize genes for functional analyses by excluding large numbers of genes from further study.  s. paratyphi c has diverse genome structures, possibly as a result of large genomic insertions, e.g., spi although the salmonella typhoid agents may not be more closely related among them than each of them to other salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes and making genome structure rearrangements. comparison of multiple salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes. 
s. paratyphi c has diverse genome structures among different isolates, possibly as a result of large genomic insertions, e.g., spi although the salmonella typhoid agents may not be more closely related among them than each of them to other salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes followed by genome structure rearrangements. comparison of multiple salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes. we constructed a high resolution genome map in s. paratyphi c strain rks and located four insertions totaling kb and seven deletions totaling kb relative to s. typhimurium lt two rearrangements were revealed, including an inversion of kb covering the ter region and the translocation of the kb i-ceui f fragment. the wild type strains analyzed in this study exhibited diverse genome structures, mostly as a result of recombination between rrn genes. in at least two cases, the rearrangements involved recombination between genomic sites other than the rrn genes, possibly homologous genes in prophages. as many but not all host-adapted salmonella pathogens have large genomic insertions as well as the supposedly resultant genomic rearrangements, bacterial genome plasticity presents an extraordinary evolutionary phenomenon. previously we reported different genome structures between two strains of s. paratyphi c, which suggests that s. paratyphi c might have a plastic genome. salmonella paratyphi c, like s. typhi, is adapted to humans and causes typhoid fever. two strains had a kb deletion between rrla and rrlb, which is a highly conservative region and no deletion has been reported in this region in any other salmonella lineages. events contributing to genomic plasticity, especially large insertions, may be associated with the formation of particular salmonella pathogens.
 salmonella paratyphi c, like s. typhi, is adapted to humans and causes typhoid fever. previously we reported different genome structures between two strains of s. paratyphi c, which suggests that s. paratyphi c might have a plastic genome. as many but not all host-adapted salmonella pathogens have large genomic insertions as well as the supposedly resultant genomic rearrangements, bacterial genome plasticity presents an extraordinary evolutionary phenomenon. events contributing to genomic plasticity, especially large insertions, may be associated with the formation of particular salmonella pathogens.  we constructed a high resolution genome map in s. paratyphi c strain rks and located four insertions totaling kb and seven deletions totaling kb relative to s. typhimurium lt two rearrangements were revealed, including an inversion of kb covering the ter region and the translocation of the kb i-ceui f fragment. the wild type strains analyzed in this study exhibited diverse genome structures, mostly as a result of recombination between rrn genes. in at least two cases, the rearrangements involved recombination between genomic sites other than the rrn genes, possibly homologous genes in prophages. two strains had a kb deletion between rrla and rrlb, which is a highly conservative region and no deletion has been reported in this region in any other salmonella lineages.  s. paratyphi c has diverse genome structures among different isolates, possibly as a result of large genomic insertions, e.g., spi although the salmonella typhoid agents may not be more closely related among them than each of them to other salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes followed by genome structure rearrangements. comparison of multiple salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes. 