BACKGROUND
the type iii secretion system  is possessed by gram-negative bacteria, especially those occurring in animal and plant pathogens, e.g.
yersinia, shigella, salmonella, pseudomonas and escherichia species  <cit> .
the t3ss secretes and translocates effector proteins into the cytosol of eukaryotic cells, thus contributing to bacterial virulence against the host  <cit> .
while the t3ss apparatus is well conserved in these bacteria, the specific properties of the effectors which are secreted via t3ss and symptomatic effects caused by the effectors on the host organism vary widely  <cit> .
vibrios are gram-negative γ-proteobacteria which are ubiquitous in marine and estuarine environments  <cit> .
several of the more than  <dig> vibrio species are pathogens for fish, shellfish, coral, and mammals  <cit> , and vibrio parahaemolyticus was the first species in which the presence of t3ss was reported  <cit> .
v. parahaemolyticus is a cause of food-borne gastroenteritis in humans, and almost all strains isolated from diarrheal patients produce the thermostable direct hemolysin  and/or the tdh-related hemolysin , which are encoded by the tdh and trh genes, respectively  <cit> .
v. parahaemolyticus strains, which exhibit the kanagawa phenomenon , a beta-hemolysis detectable on a special blood agar   <cit> , possess two tdh genes, tdha and tdhs, but not the trh gene  <cit> .
in contrast, kp-negative clinical v. parahaemolyticus strains possess the trh gene only or both the trh and tdh genes.
genome sequencing of the kp-positive v. parahaemolyticus strain rimd <dig> demonstrated that it possesses two sets of the genes for t3ss on chromosomes  <dig> and  <dig>   <cit> .
it has further been demonstrated that t3ss <dig> is involved in enterotoxicity of the organism, and is considered to be an important factor in the pathogenicity of diarrheal illness  <cit> .
the t3ss <dig> genes are located on a pathogenicity island  known as vp-pai on chromosome  <dig>  and the genes were found in kp-positive, but not in kp-negative strains  <cit> .
after the discovery of the t3ss genes in v. parahaemolyticus, other vibrios such as v. alginolyticus, v. harveyi, v. tubiashii and v. cholerae were also found to possess the genes for t3ss  <cit> .
while the t3sss of v. alginolyticus, v. harveyi and v. tubiashii, are more closely related to t3ss <dig> of v. parahaemolyticus  <cit> , that of v. cholerae is similar to t3ss <dig> of v. parahaemolyticus  <cit> .
in addition, several studies have demonstrated that some v. cholerae non-o1/non-o <dig> serogroup strains, which do not possess the cholera toxin gene, do possess a set of t3ss genes in a pai  on their chromosome  <cit> .
it has further been suggested that the t3ss of non-o1/non-o <dig> v. cholerae is also involved in the pathogenicity of the bacterium  <cit> .
in our most recently reported study, we used the sequencing and pcr assay of the genomic dna of the th <dig> strain to detect the presence of a novel pai  in trh-positive  v. parahaemolyticus strains  <cit> .
the vp-paith <dig> was found to contain a set of genes for t3ss, and the t3ss of the th <dig> strain to be essential for the enterotoxicity of this strain  <cit> .
phylogenetic analysis indicated that the t3ss genes of th <dig> are related to that of rimd <dig>  but belong to distinct lineage, with the former known as t3ss2β and the latter as t3ss2α  <cit> .
subsequent studies showed that t3ss2α and t3ss2β are present in, respectively, kp-positive and trh-positive v. parahaemolyticus strains and are also distributed among pathogenic v. cholerae non-o1/non-o <dig> serogroup strains  <cit> .
a previous study examined the distribution of the t3ss2-related genes in vibrio species, but tested only for the presence of the t3ss2α genes and in a limited number of strains from each species  <cit> .
in this study, we re-investigated the distribution of the genes for t3ss <dig> in various vibrio species and targeted both the t3ss2α and t3ss2β genes.
RESULTS
distribution of the t3ss2-related genes in vibrio species
to analyze the distribution of the t3ss2-related genes in vibrio species other than v. parahaemolyticus, pcr assays were performed using oligonucleotide primer pairs  which target the t3ss2-related genes present in the vp-pai, i.e., vscn <dig> , vscc2n2r2s2t2u <dig>  vcrd <dig> , vopb2d <dig> , or vopclp   <cit> , for  <dig> vibrio species.
the design of the pcr primer pairs was based on the gene sequences in strains rimd <dig> or th <dig>  representing t3ss2α or t3ss2β, respectively .
we tested multiple strains of several species in the genus vibrio which are implicated as pathogenic for humans, that is, v. vulnificus , v. fluvialis , v. furnissii , v. hollisae , v. cholerae  and v. mimicus .
for other species, one strain of each was tested .
the assay demonstrated that, in addition to part of the v. cholerae strains, as previously reported, amplicons of the expected size of at least several of the t3ss <dig> genes were obtained from all of the v. hollisae strains and some of the v. mimicus strains.
however, none of the genes tested in any of the remaining  <dig> species could be amplified .
among the  <dig> non-o1/non-o <dig> v. cholerae strains isolated from patients  or environments , we obtained the amplicons of at least one gene encoding the apparatus protein of the t3ss2α genes from  <dig> strains .
in two v. cholerae strains, which constitute the pcr products of t3ss2β genes, at least six genes for the apparatus and two genes for the translocons could be amplified .
we therefore concluded that the aforementioned  <dig> v. cholerae strains were t3ss2α-positive and the two were t3ss2β-positive.
of these  <dig> t3ss2-positive strains, only one, the v. cholerae strain rimd <dig>  which possesses t3ss2α genes, was isolated from the environment.
therefore, as far as we could determine in this study, t3ss <dig> genes of v. cholerae tend to be found in clinical strains rather than in environmental isolates.
in all of the five v. hollisae strains tested, the amplicons for three genes of t3ss2α, vscn <dig>  vscr <dig> and vsct <dig>  were obtained with the pcr assay, but no other t3ss2α genes or any t3ss2β genes could be amplified.
the pcr products for vscn2r2t <dig> could be partially sequenced, which confirmed that the amplicons that could be obtained are more closely related to the t3ss2α than to the t3ss2β genes .
the pcr products of the genes for t3ss <dig> were detected in nine of  <dig> clinical or environmental v. mimicus strains.
the genes encoding the apparatus proteins of t3ss <dig>  vscn2c2r2t2u <dig> and vcrd <dig>  were amplified by pcr in all the t3ss2-positive v. mimicus strains, although the amplicons for the genes encoding effector proteins, i.e., vopclp, could not be obtained in a few of these strains .
of the nine t3ss2-positive strains, at least six genes for the apparatus proteins and two genes for the translocons of t3ss2α genes could be amplified from eight strains, while pcr amplification led to the detection in a v. mimicus strain of the amplicons of the t3ss2β genes, i.e., six genes encoding the apparatus proteins vscn2c2r2t2u <dig> and vcrd <dig>  two genes encoding the translocons vopb2d <dig>  and two genes for the regulators vtrab.
in the other six v. mimicus strains, no amplicons of the genes for either type of t3ss <dig> could be obtained .
of the nine t3ss2-positive v. mimicus strains, eight were therefore identified as t3ss2α-positive, and one as t3ss2β-positive.
these findings suggest that, in addition to their distribution in v. parahaemolyticus and v. cholerae strains, the genes for t3ss <dig> are found in v. hollisae and v. mimicus strains, although only three t3ss <dig> genes were detected in v. hollisae strains.
distribution of tdh or trh in t3ss2-positive v. cholerae and v. mimicus strains
in v. parahaemolyticus, the strains which possess the t3ss <dig> gene cluster also possess the tdh and/or trh genes  <cit> .
to examine whether the tdh or trh genes also coexist in t3ss2-possessing v. cholerae and v. mimicus strains, we performed a pcr assay using the pair of primers  which target the tdh or trh genes of v. parahaemolyticus rimd <dig> or th <dig> strains, respectively.
among the t3ss2-possessing v. mimicus strains, the tdh gene could be detected in all t3ss2α-positive strains, although no amplicons of the trh gene could be obtained in v. mimicus strains, while neither of the amplicons could be obtained in the t3ss2-positive v. cholerae strains.
to analyze the distribution of the tdh or trh genes in  <dig> t3ss2-positive v. cholerae strains, we performed an additional pcr assay using the primer set  targeting the region between a33_ <dig> and the downstream region of the v. cholerae am- <dig> strain which is homologous with tdh of v. parahaemolyticus.
pcr products could be obtained for both the t3ss2α- and t3ss2β-positive v. cholerae strains, except for the rimd <dig>  strain .
these results suggest that the tdh gene may be related to the presence of the t3ss <dig> gene cluster in v. cholerae and v. mimicus strains.
gene organization of the t3ss <dig> gene cluster in v. mimicus
the results presented here demonstrated that some v. mimicus strains possess the genes for t3ss2α or t3ss2β.
since the gene organizations of the t3ss <dig> gene cluster in the organism were completely unknown, we attempted to analyze the gene organization of the t3ss <dig> region in v. mimicus strains.
to this end, we performed pcr scanning against the genomic dna of t3ss2-positive v. mimicus strains rimd <dig>  and rimd <dig>  by using six pcr primer pairs for each strain .
in the t3ss2α-positive strain rimd <dig>  pcr products of the expected size were detected for all primer pairs , thus suggesting that the gene organization of the t3ss <dig> gene cluster in strain rimd <dig> is similar to that of the t3ss2α gene cluster in v. parahaemolyticus strain rimd <dig>  in the t3ss2β-positive strain rimd <dig>  amplicons of the expected size were obtained for five primer pairs, although the size of the product obtained for a primer pair between the vopd <dig> and vopc genes was notably larger, by approximately  <dig> kb, than that of the region of the v. parahaemolyticus th <dig> .
however, the pcr product which was amplified between vopd <dig> and vopp was the same size as that of the v. parahaemolyticus th <dig> strain.
this suggested that the gene organization of t3ss <dig> of v. mimicus rimd <dig> is similar to that of t3ss2β of v. parahaemolyticus, while a genetic element of approximately  <dig> kb, which could not be detected in the region of v. parahaemolyticus strain th <dig>  may exist in the region between the vopp and vopc genes in v. mimicus strain rimd <dig>
these findings suggest that the gene organization of the t3ss <dig> gene clusters, both t3ss2α and t3ss2β, in v. mimicus strains are basically similar to those of the v. parahaemolyticus and v. cholerae strains.
phylogenetic analysis of the t3ss2-related genes in v. mimicus
next, we analyzed the phylogeny of the t3ss <dig> genes identified in v. mimicus strains.
the purified amplicons of the genes for vscn2r2t <dig> in the t3ss2-positive v. mimicus strains were sequenced and the nucleotide sequences thus obtained were used for phylogenetic analysis.
in addition, we used the nucleotide sequences of the three t3ss <dig> genes of the two v. parahaemolyticus strains rimd <dig> and th <dig>  and the four v. cholerae strains, am- <dig>   <dig> and 623- <dig>  as well as v <dig>  identified to date.
phylogenetic trees for each of the genes were constructed with the neighbor-joining  method.
the analysis demonstrated that the pcr products of the t3ss <dig> genes in v. mimicus strains rimd <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> belong to the cluster containing the t3ss2α genes of v. parahaemolyticus strain rimd <dig> and that of v. cholerae strains am- <dig> and v <dig> .
in contrast, the amplicons obtained from the t3ss <dig> genes in the v. mimicus strain rimd <dig> were found to be closely related to the t3ss2β genes in the v. parahaemolyticus th <dig> strain and v. cholerae strains  <dig> and 623- <dig> .
these findings confirmed that, similar to the findings for v. parahaemolyticus and v. cholerae strains, the t3ss <dig> of v. mimicus strains could be classified into two phylogroups, t3ss2α and t3ss2β.
presence and absence of the genes in vpi- <dig> and vp-pai
both the t3ss <dig> gene cluster of v. parahaemolyticus and the t3ss gene cluster of v. cholerae can be found on pais  <cit> .
in v. cholerae non-o1/non-o <dig> strains am- <dig>  nrt36s, v <dig>   <dig> and 623- <dig>  the pai  contains the genes for sialic acid metabolism in addition to the genes for t3ss   <cit> .
in v. parahaemolyticus strains rimd <dig> and th <dig>  on the other hand, the homologues for sialic acid metabolism were not found in the vp-pai .
the gene compositions of the pai cassettes in v. parahaemolyticus and v. cholerae, except for the t3ss gene cluster, were thus clearly distinct.
to compare the gene organization of the pai of v. mimicus with that of the pais of v. parahaemolyticus and v. cholerae, we used additional pcr assays to determine the presence or absence of open reading frames , which occur only in the vp-pai of v. parahaemolyticus or the vpi- <dig> of v. cholerae, in t3ss2-positive v. mimicus strains.
the orfs on the pais of v. parahaemolyticus and v. cholerae strains, except for the t3ss <dig> genes, could be amplified with the primer sets that were designed by using the orf sequences on the vp-pai in v. parahaemolyticus strains rimd <dig> and th <dig> and those on vpi- <dig> in v. cholerae strains am- <dig> and  <dig> as templates against the genomic dna of nine v. mimicus t3ss2-positive strains .
some of the orfs on the vp-pai of v. parahaemolyticus strains, could be amplified in the v. mimicus strains tested, but most could not .
in contrast, most of the non-t3ss orfs on vpi- <dig> of v. cholerae could be amplified in the t3ss-positive, but not in the t3ss2-negative v. mimicus strains .
these findings suggest that the composition of the v. mimicus pais containing the t3ss genes, if present, may be more closely related to that of v. cholerae vpi- <dig> than of v. parahaemolyticus vp-pai .
cytotoxicity assay of mutant strains
previous studies have demonstrated that t3ss2s of v. parahaemolyticus rimd <dig> and th <dig> as well as v. cholerae am- <dig> contribute to the pathogenicity of these organisms  <cit> .
to determine the possible contribution of t3ss <dig> to the pathogenicity of v. mimicus strains, we compared the cytotoxicity of the wild-type and mutant strains for cultured cell lines.
t3ss-deficient mutants were constructed by disruption of the homologue of the vscn <dig> gene, which encodes an atpase of t3ss <dig>  in v. mimicus rimd <dig>  and rimd <dig>  strains.
to confirm the deletion of the vscn <dig> gene, pcr amplification using oligonucleotide primer pairs was performed .
the growth of the mutant strains in lb medium  was indistinguishable from that of the parental strains .
both v. mimicus rimd <dig> and rimd <dig> strains were cytotoxic for caco- <dig> cells at  <dig> h post-infection.
the cytotoxicity of both the t3ss2α- and t3ss2β-deficient mutant strains tended to decrease, but there were no significant differences between t3ss2α- and t3ss2β-deficient mutant strains and their parental strains .
discussion
a recent study of ours demonstrated that two lines of distinct lineage of the t3ss <dig> gene cluster, t3ss2α and t3ss2β, are present in the kp-positive and trh-positive v. parahaemolyticus strains, respectively  <cit> .
although a previously reported study using dot blot analysis could not detect the genes for t3ss <dig> in  <dig> vibrio species, the probes and pcr primers used in previous studies were designed based on the sequence information of the t3ss2α genes in v. parahaemolyticus strain rimd <dig>  <cit> .
since the t3ss2β genes cannot be detected by either pcr amplifications or comparative genomic hybridization analysis targeting the t3ss2α genes  <cit> , we re-investigated the distribution of the t3ss <dig> genes, both t3ss2α and t3ss2β, in vibrio species.
to examine the distribution of the genes for t3ss <dig> in vibrios other than v. parahaemolyticus, we performed a pcr assay using pcr primer pairs targeting both the t3ss2α and t3ss2β genes.
of the  <dig> vibrio species tested, the t3ss2-related genes were detected in three species, v. cholerae, which was previously reported, as well as v. hollisae and v. mimicus.
in v. hollisae strains, only three genes for t3ss2α, vscn <dig>  vscr <dig>  and vsct <dig>  were detected.
nevertheless, the fact that the pcr reactions for these three genes were positive in all the five v. hollisae strains tested is intriguing.
we speculate that the other genes for t3ss2α might be absent in these particular v. hollisae strains, or that the sequences of the other genes included variations that would make pcr amplification with the primer pairs used in this assay difficult.
these possibilities should be examined in the future by more detailed genetic analyses, e.g.
sequencing of the region flanking the t3ss2-related genes.
a previous study showed that the t3ss2-related genes are present in v. mimicus strains  <cit> .
in our study, the pcr assay also demonstrated the presence of the t3ss <dig> genes in v. mimicus strains.
of the  <dig> v. mimicus strains tested in this study,  <dig> strains were isolated from patients and three from the environment, with all t3ss2-positive strains isolated from patients.
there were eight t3ss2α-positive and one t3ss2β-positive strain among the t3ss2-positive v. mimicus strains.
the gene organization of the t3ss <dig> gene cluster in the v. mimicus strains containing t3ss2α or t3ss2β, was basically similar to that of the v. parahaemolyticus and v. cholerae strains.
ours is thus the first study to demonstrate that the two distinct types of t3ss <dig> gene clusters, t3ss2α and t3ss2β, are present not only in v. parahaemolyticus and v. cholerae but also in v. mimicus strains.
furthermore, we could show that the structures of the v. mimicus pais containing the t3ss genes may be more closely related to those of v. cholerae vpi- <dig> than of v. parahaemolyticus vp-pai .
in contrast, the orfs in vpi- <dig> were not detected in any of the t3ss-negative v. mimicus strains.
this implies, therefore, that the similar pai cassettes containing the t3ss <dig> gene cluster were acquired through horizontal gene transfer in v. cholerae and v. mimicus .
the pcr primer pairs used in this study were found to be useful for detecting as well as distinguishing the genes for t3ss2α and t3ss2β in vibrio species.
in particular, the pcr assays targeting the three genes, vscn <dig>  vscr <dig> and vsct <dig>  produced stable and reliable results for detection of t3ss2-related genes.
we therefore consider that, for determining the presence or absence of these genes, pcr amplification using the primer pairs for the vscn2r2t <dig> genes of t3ss2α or t3ss2β is effective and rapid.
although only a limited number of strains of the non-human pathogenic vibrio species was examined in this study, more extensive studies of those species using more strains may well reveal the presence of the t3ss <dig> genes in vibrios other than the ones reported here.
previous studies showed that the t3sss of v. parahaemolyticus and v. cholerae contribute to their pathogenicity for humans  <cit> .
in v. mimicus, a bacterium which is known to be a causative agent of gastroenteritis in humans, the hemolysin was previously reported as a major virulence factor  <cit> .
to assess the function of t3ss of v. mimicus in pathogenicity in our study, we evaluated the cytotoxicity of v. mimicus for caco- <dig> cells because v. parahaemolyticus had a t3ss2-dependent cytotoxic effect on caco- <dig> cells  <cit> .
the results indicated that both t3ss2α-possessing and t3ss2β-possessing v. mimicus strains showed the cytotoxic activity on caco- <dig> cells in this assay.
although we could not detect statistically significant differences between t3ss-deficient mutants and parental strains, there was a tendency for the cytotoxicity of t3ss-deficient mutants to diminish than that of the parental mutants.
a previous report showed that the deletion of the hemolysin gene in v. mimicus significantly reduced fluid accumulation in rabbit ileal loop tests, but the mutant partially retained this action, which suggests that, besides the hemolysin, v. mimicus may contain an additional virulence determinant  <cit> .
it is therefore possible that t3ss is a candidate for the previously unidentified virulence determinant in pathogenic v. mimicus strains for humans.
the observed ambiguous differences in cytotoxicity between the mutants and the parental strains may be due to insufficient expression of t3ss of v. mimicus under the culturing conditions used in this study, because it is still unclear what the optimal conditions are for inducing t3ss of v. mimicus.
this possibility needs to be examined in future studies.
CONCLUSIONS
this study demonstrated the presence of the gene cluster for t3ss2α or t3ss2β in v. mimicus, a bacterium which is known to be a causative agent of gastroenteritis in humans.
since it was reported that the t3sss of v. parahaemolyticus and v. cholerae contribute to their pathogenicity for humans, the t3ss in v. mimicus identified in this study also might be a candidate virulence factor of this organism for humans.
this possibility needs to be examined in future studies.

 the type iii secretion system is possessed by gram-negative bacteria, especially those occurring in animal and plant pathogens, e.g. yersinia, shigella, salmonella, pseudomonas and escherichia species. the t3ss secretes and translocates effector proteins into the cytosol of eukaryotic cells, thus contributing to bacterial virulence against the host. while the t3ss apparatus is well conserved in these bacteria, the specific properties of the effectors which are secreted via t3ss and symptomatic effects caused by the effectors on the host organism vary widely. vibrios are gram-negative γ-proteobacteria which are ubiquitous in marine and estuarine environments. several of the more than vibrio species are pathogens for fish, shellfish, coral, and mammals, and vibrio parahaemolyticus was the first species in which the presence of t3ss was reported. v. parahaemolyticus is a cause of food-borne gastroenteritis in humans, and almost all strains isolated from diarrheal patients produce the thermostable direct hemolysin and/or the tdh-related hemolysin, which are encoded by the tdh and trh genes, respectively. v. parahaemolyticus strains, which exhibit the kanagawa phenomenon, a beta-hemolysis detectable on a special blood agar, possess two tdh genes, tdha and tdhs, but not the trh gene. in contrast, kp-negative clinical v. parahaemolyticus strains possess the trh gene only or both the trh and tdh genes. genome sequencing of the kp-positive v. parahaemolyticus strain rimd demonstrated that it possesses two sets of the genes for t3ss on chromosomes and. it has further been demonstrated that t3ss is involved in enterotoxicity of the organism, and is considered to be an important factor in the pathogenicity of diarrheal illness. the t3ss genes are located on a pathogenicity island known as vp-pai on chromosome and the genes were found in kp-positive, but not in kp-negative strains. after the discovery of the t3ss genes in v. parahaemolyticus, other vibrios such as v. alginolyticus, v. harveyi, v. tubiashii and v. cholerae were also found to possess the genes for t3ss. while the t3sss of v. alginolyticus, v. harveyi and v. tubiashii, are more closely related to t3ss of v. parahaemolyticus, that of v. cholerae is similar to t3ss of v. parahaemolyticus. in addition, several studies have demonstrated that some v. cholerae non-o1/non-o serogroup strains, which do not possess the cholera toxin gene, do possess a set of t3ss genes in a pai on their chromosome. it has further been suggested that the t3ss of non-o1/non-o v. cholerae is also involved in the pathogenicity of the bacterium. in our most recently reported study, we used the sequencing and pcr assay of the genomic dna of the th strain to detect the presence of a novel pai in trh-positive v. parahaemolyticus strains. the vp-paith was found to contain a set of genes for t3ss, and the t3ss of the th strain to be essential for the enterotoxicity of this strain. phylogenetic analysis indicated that the t3ss genes of th are related to that of rimd but belong to distinct lineage, with the former known as t3ss2β and the latter as t3ss2α. subsequent studies showed that t3ss2α and t3ss2β are present in, respectively, kp-positive and trh-positive v. parahaemolyticus strains and are also distributed among pathogenic v. cholerae non-o1/non-o serogroup strains. a previous study examined the distribution of the t3ss2-related genes in vibrio species, but tested only for the presence of the t3ss2α genes and in a limited number of strains from each species. in this study, we re-investigated the distribution of the genes for t3ss in various vibrio species and targeted both the t3ss2α and t3ss2β genes.  distribution of the t3ss2-related genes in vibrio species to analyze the distribution of the t3ss2-related genes in vibrio species other than v. parahaemolyticus, pcr assays were performed using oligonucleotide primer pairs which target the t3ss2-related genes present in the vp-pai, i.e., vscn, vscc2n2r2s2t2u vcrd, vopb2d, or vopclp, for vibrio species. the design of the pcr primer pairs was based on the gene sequences in strains rimd or th representing t3ss2α or t3ss2β, respectively. we tested multiple strains of several species in the genus vibrio which are implicated as pathogenic for humans, that is, v. vulnificus, v. fluvialis, v. furnissii, v. hollisae, v. cholerae and v. mimicus. for other species, one strain of each was tested. the assay demonstrated that, in addition to part of the v. cholerae strains, as previously reported, amplicons of the expected size of at least several of the t3ss genes were obtained from all of the v. hollisae strains and some of the v. mimicus strains. however, none of the genes tested in any of the remaining species could be amplified. among the non-o1/non-o v. cholerae strains isolated from patients or environments, we obtained the amplicons of at least one gene encoding the apparatus protein of the t3ss2α genes from strains. in two v. cholerae strains, which constitute the pcr products of t3ss2β genes, at least six genes for the apparatus and two genes for the translocons could be amplified. we therefore concluded that the aforementioned v. cholerae strains were t3ss2α-positive and the two were t3ss2β-positive. of these t3ss2-positive strains, only one, the v. cholerae strain rimd which possesses t3ss2α genes, was isolated from the environment. therefore, as far as we could determine in this study, t3ss genes of v. cholerae tend to be found in clinical strains rather than in environmental isolates. in all of the five v. hollisae strains tested, the amplicons for three genes of t3ss2α, vscn vscr and vsct were obtained with the pcr assay, but no other t3ss2α genes or any t3ss2β genes could be amplified. the pcr products for vscn2r2t could be partially sequenced, which confirmed that the amplicons that could be obtained are more closely related to the t3ss2α than to the t3ss2β genes. the pcr products of the genes for t3ss were detected in nine of clinical or environmental v. mimicus strains. the genes encoding the apparatus proteins of t3ss vscn2c2r2t2u and vcrd were amplified by pcr in all the t3ss2-positive v. mimicus strains, although the amplicons for the genes encoding effector proteins, i.e., vopclp, could not be obtained in a few of these strains. of the nine t3ss2-positive strains, at least six genes for the apparatus proteins and two genes for the translocons of t3ss2α genes could be amplified from eight strains, while pcr amplification led to the detection in a v. mimicus strain of the amplicons of the t3ss2β genes, i.e., six genes encoding the apparatus proteins vscn2c2r2t2u and vcrd two genes encoding the translocons vopb2d and two genes for the regulators vtrab. in the other six v. mimicus strains, no amplicons of the genes for either type of t3ss could be obtained. of the nine t3ss2-positive v. mimicus strains, eight were therefore identified as t3ss2α-positive, and one as t3ss2β-positive. these findings suggest that, in addition to their distribution in v. parahaemolyticus and v. cholerae strains, the genes for t3ss are found in v. hollisae and v. mimicus strains, although only three t3ss genes were detected in v. hollisae strains. distribution of tdh or trh in t3ss2-positive v. cholerae and v. mimicus strains in v. parahaemolyticus, the strains which possess the t3ss gene cluster also possess the tdh and/or trh genes. to examine whether the tdh or trh genes also coexist in t3ss2-possessing v. cholerae and v. mimicus strains, we performed a pcr assay using the pair of primers which target the tdh or trh genes of v. parahaemolyticus rimd or th strains, respectively. among the t3ss2-possessing v. mimicus strains, the tdh gene could be detected in all t3ss2α-positive strains, although no amplicons of the trh gene could be obtained in v. mimicus strains, while neither of the amplicons could be obtained in the t3ss2-positive v. cholerae strains. to analyze the distribution of the tdh or trh genes in t3ss2-positive v. cholerae strains, we performed an additional pcr assay using the primer set targeting the region between a33_ and the downstream region of the v. cholerae am- strain which is homologous with tdh of v. parahaemolyticus. pcr products could be obtained for both the t3ss2α- and t3ss2β-positive v. cholerae strains, except for the rimd strain. these results suggest that the tdh gene may be related to the presence of the t3ss gene cluster in v. cholerae and v. mimicus strains. gene organization of the t3ss gene cluster in v. mimicus the results presented here demonstrated that some v. mimicus strains possess the genes for t3ss2α or t3ss2β. since the gene organizations of the t3ss gene cluster in the organism were completely unknown, we attempted to analyze the gene organization of the t3ss region in v. mimicus strains. to this end, we performed pcr scanning against the genomic dna of t3ss2-positive v. mimicus strains rimd and rimd by using six pcr primer pairs for each strain. in the t3ss2α-positive strain rimd pcr products of the expected size were detected for all primer pairs, thus suggesting that the gene organization of the t3ss gene cluster in strain rimd is similar to that of the t3ss2α gene cluster in v. parahaemolyticus strain rimd in the t3ss2β-positive strain rimd amplicons of the expected size were obtained for five primer pairs, although the size of the product obtained for a primer pair between the vopd and vopc genes was notably larger, by approximately kb, than that of the region of the v. parahaemolyticus th. however, the pcr product which was amplified between vopd and vopp was the same size as that of the v. parahaemolyticus th strain. this suggested that the gene organization of t3ss of v. mimicus rimd is similar to that of t3ss2β of v. parahaemolyticus, while a genetic element of approximately kb, which could not be detected in the region of v. parahaemolyticus strain th may exist in the region between the vopp and vopc genes in v. mimicus strain rimd these findings suggest that the gene organization of the t3ss gene clusters, both t3ss2α and t3ss2β, in v. mimicus strains are basically similar to those of the v. parahaemolyticus and v. cholerae strains. phylogenetic analysis of the t3ss2-related genes in v. mimicus next, we analyzed the phylogeny of the t3ss genes identified in v. mimicus strains. the purified amplicons of the genes for vscn2r2t in the t3ss2-positive v. mimicus strains were sequenced and the nucleotide sequences thus obtained were used for phylogenetic analysis. in addition, we used the nucleotide sequences of the three t3ss genes of the two v. parahaemolyticus strains rimd and th and the four v. cholerae strains, am- and 623- as well as v identified to date. phylogenetic trees for each of the genes were constructed with the neighbor-joining method. the analysis demonstrated that the pcr products of the t3ss genes in v. mimicus strains rimd and belong to the cluster containing the t3ss2α genes of v. parahaemolyticus strain rimd and that of v. cholerae strains am- and v. in contrast, the amplicons obtained from the t3ss genes in the v. mimicus strain rimd were found to be closely related to the t3ss2β genes in the v. parahaemolyticus th strain and v. cholerae strains and 623-. these findings confirmed that, similar to the findings for v. parahaemolyticus and v. cholerae strains, the t3ss of v. mimicus strains could be classified into two phylogroups, t3ss2α and t3ss2β. presence and absence of the genes in vpi- and vp-pai both the t3ss gene cluster of v. parahaemolyticus and the t3ss gene cluster of v. cholerae can be found on pais. in v. cholerae non-o1/non-o strains am- nrt36s, v and 623- the pai contains the genes for sialic acid metabolism in addition to the genes for t3ss. in v. parahaemolyticus strains rimd and th on the other hand, the homologues for sialic acid metabolism were not found in the vp-pai. the gene compositions of the pai cassettes in v. parahaemolyticus and v. cholerae, except for the t3ss gene cluster, were thus clearly distinct. to compare the gene organization of the pai of v. mimicus with that of the pais of v. parahaemolyticus and v. cholerae, we used additional pcr assays to determine the presence or absence of open reading frames, which occur only in the vp-pai of v. parahaemolyticus or the vpi- of v. cholerae, in t3ss2-positive v. mimicus strains. the orfs on the pais of v. parahaemolyticus and v. cholerae strains, except for the t3ss genes, could be amplified with the primer sets that were designed by using the orf sequences on the vp-pai in v. parahaemolyticus strains rimd and th and those on vpi- in v. cholerae strains am- and as templates against the genomic dna of nine v. mimicus t3ss2-positive strains. some of the orfs on the vp-pai of v. parahaemolyticus strains, could be amplified in the v. mimicus strains tested, but most could not. in contrast, most of the non-t3ss orfs on vpi- of v. cholerae could be amplified in the t3ss-positive, but not in the t3ss2-negative v. mimicus strains. these findings suggest that the composition of the v. mimicus pais containing the t3ss genes, if present, may be more closely related to that of v. cholerae vpi- than of v. parahaemolyticus vp-pai. cytotoxicity assay of mutant strains previous studies have demonstrated that t3ss2s of v. parahaemolyticus rimd and th as well as v. cholerae am- contribute to the pathogenicity of these organisms. to determine the possible contribution of t3ss to the pathogenicity of v. mimicus strains, we compared the cytotoxicity of the wild-type and mutant strains for cultured cell lines. t3ss-deficient mutants were constructed by disruption of the homologue of the vscn gene, which encodes an atpase of t3ss in v. mimicus rimd and rimd strains. to confirm the deletion of the vscn gene, pcr amplification using oligonucleotide primer pairs was performed. the growth of the mutant strains in lb medium was indistinguishable from that of the parental strains. both v. mimicus rimd and rimd strains were cytotoxic for caco- cells at h post-infection. the cytotoxicity of both the t3ss2α- and t3ss2β-deficient mutant strains tended to decrease, but there were no significant differences between t3ss2α- and t3ss2β-deficient mutant strains and their parental strains. discussion a recent study of ours demonstrated that two lines of distinct lineage of the t3ss gene cluster, t3ss2α and t3ss2β, are present in the kp-positive and trh-positive v. parahaemolyticus strains, respectively. although a previously reported study using dot blot analysis could not detect the genes for t3ss in vibrio species, the probes and pcr primers used in previous studies were designed based on the sequence information of the t3ss2α genes in v. parahaemolyticus strain rimd. since the t3ss2β genes cannot be detected by either pcr amplifications or comparative genomic hybridization analysis targeting the t3ss2α genes, we re-investigated the distribution of the t3ss genes, both t3ss2α and t3ss2β, in vibrio species. to examine the distribution of the genes for t3ss in vibrios other than v. parahaemolyticus, we performed a pcr assay using pcr primer pairs targeting both the t3ss2α and t3ss2β genes. of the vibrio species tested, the t3ss2-related genes were detected in three species, v. cholerae, which was previously reported, as well as v. hollisae and v. mimicus. in v. hollisae strains, only three genes for t3ss2α, vscn vscr and vsct were detected. nevertheless, the fact that the pcr reactions for these three genes were positive in all the five v. hollisae strains tested is intriguing. we speculate that the other genes for t3ss2α might be absent in these particular v. hollisae strains, or that the sequences of the other genes included variations that would make pcr amplification with the primer pairs used in this assay difficult. these possibilities should be examined in the future by more detailed genetic analyses, e.g. sequencing of the region flanking the t3ss2-related genes. a previous study showed that the t3ss2-related genes are present in v. mimicus strains. in our study, the pcr assay also demonstrated the presence of the t3ss genes in v. mimicus strains. of the v. mimicus strains tested in this study, strains were isolated from patients and three from the environment, with all t3ss2-positive strains isolated from patients. there were eight t3ss2α-positive and one t3ss2β-positive strain among the t3ss2-positive v. mimicus strains. the gene organization of the t3ss gene cluster in the v. mimicus strains containing t3ss2α or t3ss2β, was basically similar to that of the v. parahaemolyticus and v. cholerae strains. ours is thus the first study to demonstrate that the two distinct types of t3ss gene clusters, t3ss2α and t3ss2β, are present not only in v. parahaemolyticus and v. cholerae but also in v. mimicus strains. furthermore, we could show that the structures of the v. mimicus pais containing the t3ss genes may be more closely related to those of v. cholerae vpi- than of v. parahaemolyticus vp-pai. in contrast, the orfs in vpi- were not detected in any of the t3ss-negative v. mimicus strains. this implies, therefore, that the similar pai cassettes containing the t3ss gene cluster were acquired through horizontal gene transfer in v. cholerae and v. mimicus. the pcr primer pairs used in this study were found to be useful for detecting as well as distinguishing the genes for t3ss2α and t3ss2β in vibrio species. in particular, the pcr assays targeting the three genes, vscn vscr and vsct produced stable and reliable results for detection of t3ss2-related genes. we therefore consider that, for determining the presence or absence of these genes, pcr amplification using the primer pairs for the vscn2r2t genes of t3ss2α or t3ss2β is effective and rapid. although only a limited number of strains of the non-human pathogenic vibrio species was examined in this study, more extensive studies of those species using more strains may well reveal the presence of the t3ss genes in vibrios other than the ones reported here. previous studies showed that the t3sss of v. parahaemolyticus and v. cholerae contribute to their pathogenicity for humans. in v. mimicus, a bacterium which is known to be a causative agent of gastroenteritis in humans, the hemolysin was previously reported as a major virulence factor. to assess the function of t3ss of v. mimicus in pathogenicity in our study, we evaluated the cytotoxicity of v. mimicus for caco- cells because v. parahaemolyticus had a t3ss2-dependent cytotoxic effect on caco- cells. the results indicated that both t3ss2α-possessing and t3ss2β-possessing v. mimicus strains showed the cytotoxic activity on caco- cells in this assay. although we could not detect statistically significant differences between t3ss-deficient mutants and parental strains, there was a tendency for the cytotoxicity of t3ss-deficient mutants to diminish than that of the parental mutants. a previous report showed that the deletion of the hemolysin gene in v. mimicus significantly reduced fluid accumulation in rabbit ileal loop tests, but the mutant partially retained this action, which suggests that, besides the hemolysin, v. mimicus may contain an additional virulence determinant. it is therefore possible that t3ss is a candidate for the previously unidentified virulence determinant in pathogenic v. mimicus strains for humans. the observed ambiguous differences in cytotoxicity between the mutants and the parental strains may be due to insufficient expression of t3ss of v. mimicus under the culturing conditions used in this study, because it is still unclear what the optimal conditions are for inducing t3ss of v. mimicus. this possibility needs to be examined in future studies.  this study demonstrated the presence of the gene cluster for t3ss2α or t3ss2β in v. mimicus, a bacterium which is known to be a causative agent of gastroenteritis in humans. since it was reported that the t3sss of v. parahaemolyticus and v. cholerae contribute to their pathogenicity for humans, the t3ss in v. mimicus identified in this study also might be a candidate virulence factor of this organism for humans. this possibility needs to be examined in future studies. 
pathogenic v. parahaemolyticus strains possess two sets of genes for type iii secretion system, t3ss and t3ss the latter are critical for virulence of the organism and be classified into two distinct phylogroups, t3ss2α and t3ss2β, which are reportedly also found in pathogenic v. cholerae non-o1/non-o serogroup strains. vibrio cholerae, v. parahaemolyticus, v. vulnificus and v. mimicus, are pathogens for humans. we therefore examined the distribution of the genes for t3ss in vibrios other than v. parahaemolyticus by using a pcr assay targeting both t3ss2α and t3ss2β genes. the gene organization of the t3ss gene clusters in v. mimicus strains was fundamentally similar to that of v. parahaemolyticus and v. cholerae in both t3ss2α- and t3ss2β-possessing strains. among the vibrio species tested in our study, several t3ss2-related genes were detected in three species, v. cholerae, v. mimicus and v. hollisae, and most of the essential genes for type iii secretion were present in t3ss2-positive v. cholerae and v. mimicus strains. however, whether t3ss2-related genes are present in other vibrio species remains unclear. moreover, both v. mimicus strains possessing t3ss2α and t3ss2β were identified. this study is the first reported evidence of the presence of t3ss gene clusters in v. mimicus strains. this finding thus provides a new insight into the pathogenicity of the v. mimicus species.
 vibrios, which include more than species, are ubiquitous in marine and estuarine environments, and several of them e.g. vibrio cholerae, v. parahaemolyticus, v. vulnificus and v. mimicus, are pathogens for humans. pathogenic v. parahaemolyticus strains possess two sets of genes for type iii secretion system, t3ss and t3ss the latter are critical for virulence of the organism and be classified into two distinct phylogroups, t3ss2α and t3ss2β, which are reportedly also found in pathogenic v. cholerae non-o1/non-o serogroup strains. however, whether t3ss2-related genes are present in other vibrio species remains unclear.  we therefore examined the distribution of the genes for t3ss in vibrios other than v. parahaemolyticus by using a pcr assay targeting both t3ss2α and t3ss2β genes. among the vibrio species tested in our study, several t3ss2-related genes were detected in three species, v. cholerae, v. mimicus and v. hollisae, and most of the essential genes for type iii secretion were present in t3ss2-positive v. cholerae and v. mimicus strains. moreover, both v. mimicus strains possessing t3ss2α and t3ss2β were identified. the gene organization of the t3ss gene clusters in v. mimicus strains was fundamentally similar to that of v. parahaemolyticus and v. cholerae in both t3ss2α- and t3ss2β-possessing strains.  this study is the first reported evidence of the presence of t3ss gene clusters in v. mimicus strains. this finding thus provides a new insight into the pathogenicity of the v. mimicus species. 