BACKGROUND
thermophilic campylobacter species, primarily campylobacter jejuni and c. coli, are curved, gram-negative organisms, belonging to the ε-proteobacteria, and are the most commonly recognized cause of acute bacterial diarrhea in the western world  <cit> .
campylobacter lari is a relatively recently discovered thermophilic campylobacter species that was first isolated from mammalian and avian species, particularly seagulls of the genus larus  <cit> .
c. lari has also been shown to be a cause of clinical infection  <cit> .
in addition, an atypical group of isolates of urease-positive thermophilic campylobacter  have been isolated from the natural environment in england in  <dig>  <cit> .
thereafter, these organisms were described as a biovar or variant of c. lari  <cit> .
subsequent reports described four human isolates in france  <cit> .
some additional isolates of uptc have also been reported in northern ireland  <cit>  in the netherlands  <cit>  and in japan  <cit> .
thus, these two representative taxa, namely urease-negative  c. lari and uptc occur within the species of c. lari  <cit> .
bacterial pathogens have the ability to bind to fibronectin   <cit> .
konkel et al.
identified and cloned a gene encoding a fibronectin-binding protein  from c. jejuni  <cit> .
in c. jejuni and c. coli, the cadf virulence gene encodes a  <dig> kda outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells  <cit> .
in relation to cadf of thermophilic campylobacter other than c. jejuni and c. coli described above, cadf and outer membrane protein gene f  have been identified in c. coli rm <dig> , c. lari rm <dig>  and c. upsaliensis rm <dig> , following whole genome shotgun sequence analysis  <cit> .
however, no detailed descriptions of the cadf  gene have yet appeared for these thermophilic campylobacter strains.
in addition, no reports on the cadf  gene in c. lari organisms have yet appeared.
therefore, the aim of the present study was to clone, sequence and analyze the full-length gene encoding the fn-binding  protein  and its adjacent genetic loci from several c. lari organisms .
we also aimed to confirm the expression of the gene in the c. lari cells.
RESULTS
ta cloning, sequencing and sequence analyses of the full-length cadf gene and its adjacent genetic loci from the  <dig> isolates of c. lari
the two primer pairs  successfully amplified pcr products of approximately  <dig>  and  <dig>  , respectively, with all  <dig> isolates of c. lari employed .
following ta cloning and sequencing, the combined nucleotide and deduced amino acid sequence data from the  <dig> isolates of c. lari determined have been made accessible in the ddbj/embl/genbank, with the accession numbers indicated in table  <dig>
the combined sequences of an approximately  <dig>  kbp region encoding a partial and putative ribosomal protein si rpsi open reading frame  , a nc region downstream of the orf , a putative cadf  orf , a cla_ <dig> orf , a nc region  and a partial and putative cla_ <dig> orf  were identified with all  <dig> c. lari isolates examined.
the present sequence analyses identified the putative orf for cadf  gene to be  <dig> bp  with all  <dig> c. lari isolates  and un c. lari rm  <dig>  with regard to the cadf-like gene, the sequence commenced with an atg start codon for all isolates and terminated with a taa for  <dig> isolates and with a tga for the other three isolates .
regarding putative orfs for cadf  gene, apparent size differences occurred amongst the four thermophilic campylobacter species examined,  <dig> bp  for  <dig> c. lari isolates and c. lari rm <dig> strain,  <dig>  for c. jejuni rm <dig> and nctc <dig>   <dig>  for c. coli rm <dig>  and  <dig>  for c. upsaliensis rm <dig>  as shown in table  <dig>  although in this limited study a small number of reference strains of c. jejuni, c. coli and c. upsaliensis were examined.
probable ribosome-binding  sites, agga    <cit> , that are complementary to a highly conserved sequence of ccuccu, close to the 3' end of 16s rrna, were also identified in all the c. lari isolates examined.
orf, open reading frame; cmw, calculated molecular weights; da, daltons.
in the region upstream of the cadf-like gene, a most probable promoter consensus sequence at the - <dig> region  ) was identified at the locus between np  <dig> and  <dig> bp, with all  <dig> c. lari isolates and the c. lari rm <dig> strain.
in addition, probable - <dig> regions  upstream of the - <dig> region were also identified, in all c. lari isolates examined.
a putative orf for the cla_ <dig> gene was also estimated to be  <dig> bp with all  <dig> c. lari isolates examined .
the cla_ <dig> gene commenced with a ttg and terminated with a taa with all  <dig> c. lari isolates and the c. lari rm <dig> strain.
apparent small size differences of the putative orfs for the cla_ <dig> also occurred amongst the four thermophilic campylobacter species examined .
as shown in table  <dig>  the nucleotide sequences of the full-length cadf  structural gene from the  <dig> c. lari isolates showed  <dig> - <dig> % similarities to each other .
the nucleotide sequences of the full-length cla_ <dig> structural gene from the  <dig> c. lari isolates showed  <dig>  -  <dig> % similarities to each other .
thus, the nucleotide sequence similarities of the cadf-like gene appear to be slightly higher than those of the cla_ <dig> gene, amongst the  <dig> c. lari isolates and the c. lari rm <dig> strain examined.
moreover, the deduced amino acid sequence alignment analyses were also performed for putative orfs of the full-length cadf  gene of  <dig> c. lari isolates, as well as those of c. lari rm <dig>  c. jejuni, c. coli and c. upsaliensis strains.
the putative orfs from the  <dig> c. lari isolates showed  <dig>  -  <dig> % amino acid sequence similarities to each other, and  <dig>  -  <dig> % similarities, with those of two c. jejuni strains .
they also showed  <dig>  -  <dig> % similarities with those of other thermophilic campylobacter organisms .
thus, the putative orfs of the full-length cadf  gene from the  <dig> c. lari isolates identified in the present study are identical size  with sequence heterogeneity, at both nucleotide and amino acid levels.
as shown in table  <dig>  the deduced amino acid sequence similarities were also examined for the putative orfs of the full-length cla_ <dig> gene among the  <dig> c. lari isolates  and other thermophilic campylobacter organisms , employed as references .
thus, cadf  gene is highly conserved among c. lari organisms isolated from humans and natural environments in several countries of asia, europe and north america.
in relation to the nc regions, two nc regions of approximately  <dig> bp, including a promoter at the - <dig> region and  <dig> bp occurred upstream of the cadf  gene and downstream of the cla_ <dig> gene, respectively, when examined combined sequences from all  <dig> c. lari isolates.
nucleotide sequences of approximately  <dig> bp from the  <dig> c. lari isolates and c. lari rm <dig> showed  <dig>  -  <dig> % sequences similarities to each other .
nucleotide sequences of approximately  <dig> bp also showed  <dig>  -  <dig> % sequence similarities among the  <dig> c. lari isolates.
thus, a considerable genetic heterogeneity of nucleotide sequences in the  <dig> bp nc region, full-length cadf  gene, full-length cla_ <dig> gene and the  <dig> bp nc region identified in the present study also occurred among the  <dig> c. lari isolates including the c. lari rm <dig> strain.
nc, non-coding.
northern blot hybridization, reverse transcription-pcr and primer extension analysis
northern blot hybridization analysis detected the cadf  gene transcription in the two c. lari isolates cells, un c. lari jcm2530t and uptc cf89- <dig> .
since the positive signals of the hybridization were shown at around  <dig>  bp , the cadf  gene may possibly be transcribed together with the cla_ <dig> gene.
thus, cadf  gene transcription was confirmed in the c. lari organisms.
when rt-pcr analysis was carried out for the rna components extracted from the un c. lari jcm2530t and uptc isolates cf89- <dig> cells with the primer pair of f-cadf <dig> in the cadf  gene and r-cadf <dig> in the cla_ <dig> gene, as shown in figure  <dig>  a positive rt-pcr signal was detected at around  <dig> bp region with both isolates, respectively .
the transcription initiation site for the cadf  gene was determined by the primer extension analysis .
the + <dig> transcription initiation site for the cadf  gene is underlined in the following sequence; 5'-ttttataatttcaaag-3', as shown in figure 2c.
deduced amino acid sequence alignment analysis and phylogenetic analyses of the cadf  orf
we carried out deduced amino acid sequence alignment analysis to elucidate the differences in cadf  protein amongst the thermophilic campylobacter.
as shown in figure  <dig>  the c. coli rm <dig> strain carried a strech of  <dig> amino acid  from amino acid positions  <dig> to  <dig>  as well as a q at amino acid position  <dig>  and regarding the nine larger amino acid for c. lari isolates than c. jejuni strains, four amino acid sequences  from amino acid positions  <dig> to  <dig> and five  from  <dig> to  <dig> were identified to occur.
when, in retation to a single fn-binding domain localized at four amino acid   <cit> , amino acid sequence alignment analysis was carried out, the putative cadf  orfs from all  <dig> c. lari isolates examined showed amino acid residues of falg  within the amino acid positions 137- <dig> instead of the frls residues, as shown in figure  <dig>
a dendrogram showing phylogenetic relationships constructed by the nj method  <cit>  based on nucleotide sequence information of full-length cadf  gene from  <dig> c. lari isolates and c. lari rm <dig> and other thermophilic campylobacter reference strains, the  <dig> c. lari isolates forming a major cluster separating from the other three thermophilic campylobacter spp.
.
in addition, un c. lari and uptc organisms were not different and similar based on the nucleotide sequence data of the cadf  gene, as shown in figure  <dig>
discussion
this is the first demonstration of the structural analysis of the full-length gene encoding a cadf  protein and its adjacent genetic loci within c. lari.
regarding the nc region upstream of the cadf  gene, this region is approximately  <dig> bp in length with all  <dig> c. lari isolates and c. lari rm <dig> strain.
however, the nc regions from the eight c. jejuni and a c. coli reference strains shown in table  <dig> examined, are shorter than those and approximately  <dig> bp in length with unknown reason.
in  <dig>  koebnik described a peptidoglycan-associating alpha-helical consensus motif in the c-terminal regions of  <dig> bacterial cell-surface proteins   <cit> .
when we compared the corresponding amino acid sequences of the putative cadf  orf from the  <dig> c. lari and some c. jejuni isolates with this consensus motif, the motif was completely conserved amongst the cadf  orfs from the isolates .
as shown in table  <dig>  the cmw of the putative cadf  orf was estimated to be  <dig>  to  <dig>  da for the  <dig> c. lari isolates and c. lari rm <dig> reference strain .
in addition, the value was also estimated to be approximately  <dig> kda for the two c. jejuni reference strains .
these estimated cmw values are in agreement with the previous description of the immunodetection of the cadf protein from five c. jejuni and c. coli isolates  <cit> .
when the nucleotide and deduced amino acid sequence alignment analyses were carried out for the putative cadf  orf, apparent size differences occurred amongst the four thermophilic campylobacter species, as described above.
regarding the putative orfs for cadf  gene between c. lari and c. jejuni organisms, nine amino acid residues are shorter in c. jejuni strains than in c. lari isolates.
recently, krause-gruszczynska et al.
described that the cadf protein from c. coli strains was  <dig> amino acid larger than those from c. jejuni strains, based on the deduced amino acid sequence alignment analysis  <cit> .
this is consistent with our present results .
they also indicated that c. coli strains carried a stretch of  <dig> amino acid in the middle region of the protein  <cit> .
in addition, in the present study, the deduced cadf  protein was shown to be  <dig> amino acid from all  <dig> c. lari isolates and were nine amino acid larger than cadf from two c. jejuni strains  .
then, we carried out deduced amino acid sequence alignment analysis to elucidate the differences in cadf  protein between c. lari and c. jejuni organisms.
as shown in figure  <dig>  the c. coli rm <dig> strain carried a stretch of  <dig> amino acid  from amino acid positions  <dig> to  <dig> as well as a q at amino acid position  <dig> .
in relation to the nine larger amino acid for c. lari isolates than c. jejuni strains, interestingly, four amino acid sequences  from amino acid positions  <dig> to  <dig> and five  from  <dig> to  <dig> were identified, as shown in figure  <dig>
regarding the cadf in campylobacter, the cadf virulence gene, encoding  <dig> kda outer membrane protein that promotes the binding of the pathogens to intestinal epithelial cells, was identified and cloned  <cit> .
in relation to identification of the binding domain within c. jejuni cadf, konkel et al.
recently described that a single fn-binding domain was localized at four amino acids , consisting of the residues, phenylalanine-arginine-leucine-serine   <cit> .
however, when amino acid sequence alignment analysis was carried out, the putative cadf  orfs from all  <dig> c. lari isolates examined in the present study showed amino acid residues of falg  within the amino acid positions  <dig> -  <dig>  instead of the frls residues .
no frls residues were also detected within any other regions of the cadf  orf from all  <dig> c. lari isolates examined.
interestingly, fnlg residues within adpb  in prevotella intermedia   <cit>  was 75% identical to the falg from c. lari .
therefore, it may be important to clarify if the cadf  protein from c. lari isolates can bind to fibronectin or not.
an experiment is now in progress to resolve this.
in the present study, for the first time, we have described the cloning, sequencing and characterization of full-length cla_ <dig> from the  <dig> c. lari isolates.
the cmw values were estimated to be  <dig>  -  <dig>  da for the  <dig> c. lari isolates and c. lari rm <dig> strain and these values were also equivalent to those from two c. jejuni and a c. coli reference strains .
in addition, the cadf  gene and the cla_ <dig> gene may possibly be functional within c. lari isolates, based on the present northern blot hybridization and rt-pcr observations, as shown in figure 2a and 2b.
thus, the cadf  gene and the cla_ <dig> gene could be co-transcribed within c. lari organisms, consisting of an operon.
since the cla_ <dig> showed a high deduced amino acid sequence similarity to the escherichia coli haloacid dehalogenase-like phosphatase  <cit> , these two may have an important biological relationship within the c. lari cells.
in the present study, the authors designed two novel primer pairs  in silico for amplification of an approximate  <dig>  kbp region, including the full-length cadf  gene and its adjacent genetic loci, based on sequence information of c. lari rm <dig>  c. jejuni rm <dig> and c. coli rm <dig> strains, resulting in successful amplification, ta-cloning and sequencing of those from the  <dig> c. lari isolates isolated from differencet sources and in several countries.
therefore, the present novel pcr primer pairs would be likely of value for, c. jejuni and c. coli organisms, as well as for other c. lari isolates.
a dendrogram showing phylogenetic relationships was constructed by the nj method  <cit> , based on nucleotide sequence information of full-length cadf  gene from  <dig> c. lari isolates and c. lari rm <dig> and other thermophilic campylobacter reference strains.
as shown in figure  <dig>  the  <dig> c. lari isolates form a major cluster separating from the other three thermophilic campylobacter spp.
in addition, the  <dig> c. lari isolates form some minor clusters, respectively, based on nucleotide sequence information from cadf  gene .
thus, nucleotide sequence information of full-length cadf  gene can be regarded as reliable in the molecular discrimination of c. lari organisms from the other three thermophilic campylobacters.
in addition, figure  <dig> also indicated that nj dendrogram of un c. lari and uptc organisms were not different and similar based on the nucleotide sequence data of the cadf-like gene.
CONCLUSIONS
the combined sequences encoding a partial and putative rpsi open reading frame , non-coding  region, a putative orf for the campylobacter adhesin to fibronectin-like gene, a putative cla_ <dig> orf, nc region and a partial and putative cla_ <dig> orf, were identified in  <dig> campylobacter lari isolates, using two novel degenerate primer pairs.
transcription of the cadf-like gene in c. lari cells in vivo was also confirmed and the transcription initiation site was determined.
the putative cadf  orfs from all c. lari isolates were nine amino acid larger than those from c. jejuni, and showed amino acid residues  <dig> - <dig> of falg , instead of the frls residues of the maximal fibronectin-binding activity site demonstrated within c. jejuni cadf.

 thermophilic campylobacter species, primarily campylobacter jejuni and c. coli, are curved, gram-negative organisms, belonging to the ε-proteobacteria, and are the most commonly recognized cause of acute bacterial diarrhea in the western world. campylobacter lari is a relatively recently discovered thermophilic campylobacter species that was first isolated from mammalian and avian species, particularly seagulls of the genus larus. c. lari has also been shown to be a cause of clinical infection. in addition, an atypical group of isolates of urease-positive thermophilic campylobacter have been isolated from the natural environment in england in. thereafter, these organisms were described as a biovar or variant of c. lari. subsequent reports described four human isolates in france. some additional isolates of uptc have also been reported in northern ireland in the netherlands and in japan. thus, these two representative taxa, namely urease-negative c. lari and uptc occur within the species of c. lari. bacterial pathogens have the ability to bind to fibronectin. konkel et al. identified and cloned a gene encoding a fibronectin-binding protein from c. jejuni. in c. jejuni and c. coli, the cadf virulence gene encodes a kda outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. in relation to cadf of thermophilic campylobacter other than c. jejuni and c. coli described above, cadf and outer membrane protein gene f have been identified in c. coli rm, c. lari rm and c. upsaliensis rm, following whole genome shotgun sequence analysis. however, no detailed descriptions of the cadf gene have yet appeared for these thermophilic campylobacter strains. in addition, no reports on the cadf gene in c. lari organisms have yet appeared. therefore, the aim of the present study was to clone, sequence and analyze the full-length gene encoding the fn-binding protein and its adjacent genetic loci from several c. lari organisms. we also aimed to confirm the expression of the gene in the c. lari cells.  ta cloning, sequencing and sequence analyses of the full-length cadf gene and its adjacent genetic loci from the isolates of c. lari the two primer pairs successfully amplified pcr products of approximately and, respectively, with all isolates of c. lari employed. following ta cloning and sequencing, the combined nucleotide and deduced amino acid sequence data from the isolates of c. lari determined have been made accessible in the ddbj/embl/genbank, with the accession numbers indicated in table the combined sequences of an approximately kbp region encoding a partial and putative ribosomal protein si rpsi open reading frame, a nc region downstream of the orf, a putative cadf orf, a cla_ orf, a nc region and a partial and putative cla_ orf were identified with all c. lari isolates examined. the present sequence analyses identified the putative orf for cadf gene to be bp with all c. lari isolates and un c. lari rm with regard to the cadf-like gene, the sequence commenced with an atg start codon for all isolates and terminated with a taa for isolates and with a tga for the other three isolates. regarding putative orfs for cadf gene, apparent size differences occurred amongst the four thermophilic campylobacter species examined, bp for c. lari isolates and c. lari rm strain, for c. jejuni rm and nctc for c. coli rm and for c. upsaliensis rm as shown in table although in this limited study a small number of reference strains of c. jejuni, c. coli and c. upsaliensis were examined. probable ribosome-binding sites, agga, that are complementary to a highly conserved sequence of ccuccu, close to the 3' end of 16s rrna, were also identified in all the c. lari isolates examined. orf, open reading frame; cmw, calculated molecular weights; da, daltons. in the region upstream of the cadf-like gene, a most probable promoter consensus sequence at the - region ) was identified at the locus between np and bp, with all c. lari isolates and the c. lari rm strain. in addition, probable - regions upstream of the - region were also identified, in all c. lari isolates examined. a putative orf for the cla_ gene was also estimated to be bp with all c. lari isolates examined. the cla_ gene commenced with a ttg and terminated with a taa with all c. lari isolates and the c. lari rm strain. apparent small size differences of the putative orfs for the cla_ also occurred amongst the four thermophilic campylobacter species examined. as shown in table the nucleotide sequences of the full-length cadf structural gene from the c. lari isolates showed - % similarities to each other. the nucleotide sequences of the full-length cla_ structural gene from the c. lari isolates showed - % similarities to each other. thus, the nucleotide sequence similarities of the cadf-like gene appear to be slightly higher than those of the cla_ gene, amongst the c. lari isolates and the c. lari rm strain examined. moreover, the deduced amino acid sequence alignment analyses were also performed for putative orfs of the full-length cadf gene of c. lari isolates, as well as those of c. lari rm c. jejuni, c. coli and c. upsaliensis strains. the putative orfs from the c. lari isolates showed - % amino acid sequence similarities to each other, and - % similarities, with those of two c. jejuni strains. they also showed - % similarities with those of other thermophilic campylobacter organisms. thus, the putative orfs of the full-length cadf gene from the c. lari isolates identified in the present study are identical size with sequence heterogeneity, at both nucleotide and amino acid levels. as shown in table the deduced amino acid sequence similarities were also examined for the putative orfs of the full-length cla_ gene among the c. lari isolates and other thermophilic campylobacter organisms, employed as references. thus, cadf gene is highly conserved among c. lari organisms isolated from humans and natural environments in several countries of asia, europe and north america. in relation to the nc regions, two nc regions of approximately bp, including a promoter at the - region and bp occurred upstream of the cadf gene and downstream of the cla_ gene, respectively, when examined combined sequences from all c. lari isolates. nucleotide sequences of approximately bp from the c. lari isolates and c. lari rm showed - % sequences similarities to each other. nucleotide sequences of approximately bp also showed - % sequence similarities among the c. lari isolates. thus, a considerable genetic heterogeneity of nucleotide sequences in the bp nc region, full-length cadf gene, full-length cla_ gene and the bp nc region identified in the present study also occurred among the c. lari isolates including the c. lari rm strain. nc, non-coding. northern blot hybridization, reverse transcription-pcr and primer extension analysis northern blot hybridization analysis detected the cadf gene transcription in the two c. lari isolates cells, un c. lari jcm2530t and uptc cf89-. since the positive signals of the hybridization were shown at around bp, the cadf gene may possibly be transcribed together with the cla_ gene. thus, cadf gene transcription was confirmed in the c. lari organisms. when rt-pcr analysis was carried out for the rna components extracted from the un c. lari jcm2530t and uptc isolates cf89- cells with the primer pair of f-cadf in the cadf gene and r-cadf in the cla_ gene, as shown in figure a positive rt-pcr signal was detected at around bp region with both isolates, respectively. the transcription initiation site for the cadf gene was determined by the primer extension analysis. the + transcription initiation site for the cadf gene is underlined in the following sequence; 5'-ttttataatttcaaag-3', as shown in figure 2c. deduced amino acid sequence alignment analysis and phylogenetic analyses of the cadf orf we carried out deduced amino acid sequence alignment analysis to elucidate the differences in cadf protein amongst the thermophilic campylobacter. as shown in figure the c. coli rm strain carried a strech of amino acid from amino acid positions to as well as a q at amino acid position and regarding the nine larger amino acid for c. lari isolates than c. jejuni strains, four amino acid sequences from amino acid positions to and five from to were identified to occur. when, in retation to a single fn-binding domain localized at four amino acid, amino acid sequence alignment analysis was carried out, the putative cadf orfs from all c. lari isolates examined showed amino acid residues of falg within the amino acid positions 137- instead of the frls residues, as shown in figure a dendrogram showing phylogenetic relationships constructed by the nj method based on nucleotide sequence information of full-length cadf gene from c. lari isolates and c. lari rm and other thermophilic campylobacter reference strains, the c. lari isolates forming a major cluster separating from the other three thermophilic campylobacter spp.. in addition, un c. lari and uptc organisms were not different and similar based on the nucleotide sequence data of the cadf gene, as shown in figure discussion this is the first demonstration of the structural analysis of the full-length gene encoding a cadf protein and its adjacent genetic loci within c. lari. regarding the nc region upstream of the cadf gene, this region is approximately bp in length with all c. lari isolates and c. lari rm strain. however, the nc regions from the eight c. jejuni and a c. coli reference strains shown in table examined, are shorter than those and approximately bp in length with unknown reason. in koebnik described a peptidoglycan-associating alpha-helical consensus motif in the c-terminal regions of bacterial cell-surface proteins. when we compared the corresponding amino acid sequences of the putative cadf orf from the c. lari and some c. jejuni isolates with this consensus motif, the motif was completely conserved amongst the cadf orfs from the isolates. as shown in table the cmw of the putative cadf orf was estimated to be to da for the c. lari isolates and c. lari rm reference strain. in addition, the value was also estimated to be approximately kda for the two c. jejuni reference strains. these estimated cmw values are in agreement with the previous description of the immunodetection of the cadf protein from five c. jejuni and c. coli isolates. when the nucleotide and deduced amino acid sequence alignment analyses were carried out for the putative cadf orf, apparent size differences occurred amongst the four thermophilic campylobacter species, as described above. regarding the putative orfs for cadf gene between c. lari and c. jejuni organisms, nine amino acid residues are shorter in c. jejuni strains than in c. lari isolates. recently, krause-gruszczynska et al. described that the cadf protein from c. coli strains was amino acid larger than those from c. jejuni strains, based on the deduced amino acid sequence alignment analysis. this is consistent with our present results. they also indicated that c. coli strains carried a stretch of amino acid in the middle region of the protein. in addition, in the present study, the deduced cadf protein was shown to be amino acid from all c. lari isolates and were nine amino acid larger than cadf from two c. jejuni strains. then, we carried out deduced amino acid sequence alignment analysis to elucidate the differences in cadf protein between c. lari and c. jejuni organisms. as shown in figure the c. coli rm strain carried a stretch of amino acid from amino acid positions to as well as a q at amino acid position. in relation to the nine larger amino acid for c. lari isolates than c. jejuni strains, interestingly, four amino acid sequences from amino acid positions to and five from to were identified, as shown in figure regarding the cadf in campylobacter, the cadf virulence gene, encoding kda outer membrane protein that promotes the binding of the pathogens to intestinal epithelial cells, was identified and cloned. in relation to identification of the binding domain within c. jejuni cadf, konkel et al. recently described that a single fn-binding domain was localized at four amino acids, consisting of the residues, phenylalanine-arginine-leucine-serine. however, when amino acid sequence alignment analysis was carried out, the putative cadf orfs from all c. lari isolates examined in the present study showed amino acid residues of falg within the amino acid positions - instead of the frls residues. no frls residues were also detected within any other regions of the cadf orf from all c. lari isolates examined. interestingly, fnlg residues within adpb in prevotella intermedia was 75% identical to the falg from c. lari. therefore, it may be important to clarify if the cadf protein from c. lari isolates can bind to fibronectin or not. an experiment is now in progress to resolve this. in the present study, for the first time, we have described the cloning, sequencing and characterization of full-length cla_ from the c. lari isolates. the cmw values were estimated to be - da for the c. lari isolates and c. lari rm strain and these values were also equivalent to those from two c. jejuni and a c. coli reference strains. in addition, the cadf gene and the cla_ gene may possibly be functional within c. lari isolates, based on the present northern blot hybridization and rt-pcr observations, as shown in figure 2a and 2b. thus, the cadf gene and the cla_ gene could be co-transcribed within c. lari organisms, consisting of an operon. since the cla_ showed a high deduced amino acid sequence similarity to the escherichia coli haloacid dehalogenase-like phosphatase, these two may have an important biological relationship within the c. lari cells. in the present study, the authors designed two novel primer pairs in silico for amplification of an approximate kbp region, including the full-length cadf gene and its adjacent genetic loci, based on sequence information of c. lari rm c. jejuni rm and c. coli rm strains, resulting in successful amplification, ta-cloning and sequencing of those from the c. lari isolates isolated from differencet sources and in several countries. therefore, the present novel pcr primer pairs would be likely of value for, c. jejuni and c. coli organisms, as well as for other c. lari isolates. a dendrogram showing phylogenetic relationships was constructed by the nj method, based on nucleotide sequence information of full-length cadf gene from c. lari isolates and c. lari rm and other thermophilic campylobacter reference strains. as shown in figure the c. lari isolates form a major cluster separating from the other three thermophilic campylobacter spp. in addition, the c. lari isolates form some minor clusters, respectively, based on nucleotide sequence information from cadf gene. thus, nucleotide sequence information of full-length cadf gene can be regarded as reliable in the molecular discrimination of c. lari organisms from the other three thermophilic campylobacters. in addition, figure also indicated that nj dendrogram of un c. lari and uptc organisms were not different and similar based on the nucleotide sequence data of the cadf-like gene.  the combined sequences encoding a partial and putative rpsi open reading frame, non-coding region, a putative orf for the campylobacter adhesin to fibronectin-like gene, a putative cla_ orf, nc region and a partial and putative cla_ orf, were identified in campylobacter lari isolates, using two novel degenerate primer pairs. transcription of the cadf-like gene in c. lari cells in vivo was also confirmed and the transcription initiation site was determined. the putative cadf orfs from all c. lari isolates were nine amino acid larger than those from c. jejuni, and showed amino acid residues - of falg, instead of the frls residues of the maximal fibronectin-binding activity site demonstrated within c. jejuni cadf. 
a neighbor joining tree constructed based on cadf gene sequence information formed a major cluster consisting of c. lari isolates, separating from the other three thermophilic campylobacters. the putative cadf orfs from all c. lari isolates were nine amino acid larger than those from c. jejuni, and showed amino acid residues - of falg, instead of the frls residues of the maximal fibronectin-binding activity site demonstrated within c. jejuni cadf. the combined sequences encoding a partial and putative rpsi open reading frame, non-coding region, a putative orf for the campylobacter adhesin to fibronectin-like protein, a putative cla_ orf, nc region and a partial and putative cla_ orf, were identified in campylobacter lari isolates, using two novel degenerate primer pairs. thus, cadf gene is highly conserved among c. lari organisms. a peptidoglycan-associating alpha-helical motif in the c-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadf orfs from the c. lari isolates. probable consensus sequence at the - and - regions were identified in all c. lari isolates, as a promoter. transcription of the cadf gene in c. lari cells in vivo was also confirmed and the transcription initiation site was determined.
 the combined sequences encoding a partial and putative rpsi open reading frame, non-coding region, a putative orf for the campylobacter adhesin to fibronectin-like protein, a putative cla_ orf, nc region and a partial and putative cla_ orf, were identified in campylobacter lari isolates, using two novel degenerate primer pairs. probable consensus sequence at the - and - regions were identified in all c. lari isolates, as a promoter.  thus, cadf gene is highly conserved among c. lari organisms. transcription of the cadf gene in c. lari cells in vivo was also confirmed and the transcription initiation site was determined. a peptidoglycan-associating alpha-helical motif in the c-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadf orfs from the c. lari isolates.  the putative cadf orfs from all c. lari isolates were nine amino acid larger than those from c. jejuni, and showed amino acid residues - of falg, instead of the frls residues of the maximal fibronectin-binding activity site demonstrated within c. jejuni cadf. a neighbor joining tree constructed based on cadf gene sequence information formed a major cluster consisting of c. lari isolates, separating from the other three thermophilic campylobacters. 