Genetic variants of Kudoa septempunctata (Myxozoa: Multivalvulida), a flounder parasite causing foodborne disease.

Foodborne disease outbreaks caused by raw olive flounders (Paralichthys olivaceus) parasitized with Kudoa septempunctata have been reported in Japan. Origins of olive flounders consumed in Japan vary, being either domestic or imported, and aquaculture-raised or natural. Although it is unknown whether different sources are associated with different outcomes, it is desirable to identify whether this is the case by determining whether unique K. septempunctata strains occur and if so, whether some are associated with foodborne illness. We here developed an intraspecific genotyping method, using the sequence variation of mitochondrial genes. We collected olive flounder samples from foodborne disease outbreaks, domestic fish farms or quarantine offices and investigated whether K. septempunctata genotype is associated with pathogenicity or geographic origin. The 104 samples were classified into three genotypes, ST1, ST2 and ST3. Frequency of symptomatic cases differed by genotypes, but the association was not statistically significant. Whereas K. septempunctata detected from aquaculture-raised and natural fish from Japan were either ST1 or ST2, those from fish inspected at quarantine from Korea to Japan were ST3. Our method can be applied to phylogeographic analysis of K. septempunctata and contribute to containing the foodborne disease. The genotype database is hosted in the PubMLST website (http://pubmlst.org/kseptempunctata/).

Characteristic mutations found in the ML0411 gene of Mycobacterium leprae isolated in Northeast Asian countries.

Genome analysis of Mycobacterium leprae strain Kyoto-2 in this study revealed characteristic nucleotide substitutions in gene ML0411, compared to the reference genome M. leprae strain TN. The ML0411 gene of Kyoto-2 had six SNPs compared to that of TN. All SNPs in ML0411 were non-synonymous mutations that result in amino acid replacements. In addition, a seventh SNP was found 41 bp upstream of the start codon in the regulatory region. The seven SNP sites in the ML0411 region were investigated by sequencing in 36 M. leprae isolates from the Leprosy Research Center in Japan. The SNP pattern in 14 of the 36 isolates showed similarity to that of Kyoto-2. Determination of the standard SNP types within the 36 stocked isolates revealed that almost all of the Japanese strains belonged to SNP type III, with nucleotide substitutions at position 14676, 164275, and 2935685 of the M. leprae TN genome. The geographical distribution pattern of east Asian M. leprae isolates by discrimination of ML0411 SNPs was investigated and interestingly turned out to be similar to that of tandem repeat numbers of GACATC in the rpoT gene (3 copies or 4 copies), which has been established as a tool for M. leprae genotyping. All seven Korean M. leprae isolates examined in this study, as well as those derived from Honshu Island of Japan, showed 4 copies of the 6-base tandem repeat plus the ML0411 SNPs observed in M. leprae Kyoto-2. They are termed Northeast Asian (NA) strain of M. leprae. On the other hand, many of isolates derived from the Okinawa Islands of Japan and from the Philippines showed 3 copies of the 6-base tandem repeat in addition to the M. leprae TN ML0411 type of SNPs. These results demonstrate the existence of M. leprae strains in Northeast Asian region having characteristic SNP patterns.

Molecular analysis and characterisation of the full-length flagellin C gene (flaC) from Campylobacter lari.

A degenerate polymerase chain reaction (PCR) primer pair (f-ClflaC/r-ClflaC) was constructed in silico to amplify flaC and its adjacent genetic loci from Campylobacter lari isolates. Approximately 1.45 kbp amplicons, including the sequences encoding the flaC structural gene of 750 bp, putative promoter, rho-independent intrinsic terminator regions and partial sequences of two putative open reading frames (ORFs), immediately upstream and downstream of the gene, were identified in 16 C. lari isolates (four urease-negative [UN] C. lari; 12 urease-positive thermophilic campylobacters [UPTC)]). All 16 flaC structural genes commenced with an ATG start codon and terminated with a TAA stop codon and probable ribosome-binding sites were identified in all 16 isolates. These probably indicate a monocistronic operon structure for the flaC gene in C. lari isolates. In addition, the putative flaC gene ORFs were deduced to be similar in 747 bp among all 26 thermophilic Campylobacter isolates examined, resulting in a similar calculated molecular weight of approximately 26.6-26.9 kDa. The flaC from C. lari was different from the flaA-like sequence and the shorter flaA of UPTC isolates found previously. Reverse transcription PCR and Northern blot hybridisation analyses identified flaC transcription in C. lari cells. The transcription initiation site for the flaC gene was also determined by primer extension analysis. A dendrogram constructed, based on the nucleotide sequence information of flaC from 17 C. lari isolates, demonstrated that the C. lari isolates were genetically variable and formed two minor clusters for UN C. lari and UPTC.

Structural analysis and expression of the full-length cytochrome P450 gene operon in Campylobacter lari.

Two sets of PCR primers are constructed to clone the cytochrome P450 structural gene, including putative promoter and terminator structures, and its adjacent genetic loci in Campylobacter lari isolates. The putative open reading frames (ORFs) of the P450 genes from 11 C. lari isolates (n=5 for urease-negative (UN) C. lari; n=6 urease-positive thermophilic campylobacters [UPTC]) examined consisted of 1365 or 1371 bases (455 or 457 amino acid residues), differing from those of the other thermophilic campylobacters (1359 [453] for C. jejuni and C. upsaliensis; 1368 [456] for C. coli). Each of the putative ORFs from the 11 isolates examined was also shown to carry start and stop codons and ribosome binding sites. Two putative promoter structures, consisting of sequences at the -35- and -10-like regions were also identified upstream of the ORFs. A single copy of the P450 gene in the genome was identified with UN C. lari JCM2530(T) and UPTC CF89-12, based on Southern blot hybridisation analysis. In addition, when reverse transcription polymerase chain reaction (RT-PCR) analyses were carried out, the transcription of the P450 structural gene in C. lari organisms in vivo was confirmed. The transcription initiation site for the gene was also determined. High nucleotide sequence similarities (95.2-98.8%) of the full-length P450 structural gene were shown with each of the 12 C. lari isolates. The UN C. lari and UPTC organisms showed similar findings with the neighbour-joining method, based on the sequence information of the P450 structural gene.

Campylobacter lari: molecular and comparative analyses of the virulence-associated chromosome locus J (vacJ) gene homologue, including the promoter region.

Following TA cloning and sequencing with a novel in silico-designed polymerase chain reaction (PCR) primer pair (f-ClvacJ/r-ClvacJ), approximately 750 base pairs (bp) of promoter and structural gene regions for vacJ and its adjacent genetic loci (approximately 1.14 kbp) were identified in 20 isolates of Campylobacter lari (urease-negative C. lari [n=7]; urease-positive thermophilic Campylobacter [n=13]). The nucleotide sequences of an approximately 70-bp non-coding region, including the typical promoter structure, showed sequence differences at 12 loci among 21 isolates including C. lari RM2100. The putative sigma70 promoter region upstream of the putative open reading frame (ORF), a start codon TTG and a probable ribosome binding site, AGGA, for the vacJ gene were also identified in all 21 C. lari isolates examined. Each ORF for the vacJ terminated with a TAA stop codon. No hypothetical transcriptional terminators were identified within the amplicons. The putative ORFs of the vacJ gene from 21 C. lari isolates consisted of 684 bases, similarly differing from those of the other thermophilic campylobacters (696 bases for C. jejuni RM1221 and NCTC11168 and C. coli RM2228; 690 for C. upsaliensis RM3195). Reverse transcription PCR analysis confirmed the transcription of the vacJ gene in the C. lari cells. A neighbour joining tree suggested a strong molecular discrimination efficacy between UPTC and UN C. lari employing vacJ nucleotide sequence information. The vacJ gene homologue from C. lari organisms appears not to be a lipoprotein signal peptide or a signal peptide in silico.

Genetic heterogeneity of the dnaK gene locus including transcription terminator region (TTR) in Campylobacter lari.

Nucleotide sequences of approximately 3.1 kbp consisting of the full-length open reading frame (ORF) for grpE, a non-coding (NC) region and a putative ORF for the full-length dnaK gene (1860 bp) were identified from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 isolate. Then, following the construction of a new degenerate polymerase chain reaction (PCR) primer pair for amplification of the dnaK structural gene, including the transcription terminator region of C. lari isolates, the dnaK region was amplified successfully, TA-cloned and sequenced in nine C. lari isolates. The dnaK gene sequences commenced with an ATG and terminated with a TAA in all 10 isolates, including CF89-12. In addition, the putative ORFs for the dnaK gene locus from seven UPTC isolates consisted of 1860 bases, and the four urease-negative (UN) C. lari isolates included C. lari RM2100 reference strain 1866. Interestingly, different probable ribosome binding sites and hypothetically intrinsic p-independent terminator structures were identified between the seven UPTC and four UN C. lari isolates, respectively. Moreover, it is interesting to note that 20 out of a total of 28 polymorphic sites occurred among amino acid sequences of the dnaK ORF from 11 C. lari isolates, identified to be alternatively UPTC-specific or UN C. lari-specific. In the neighbour-joining tree based on the nucleotide sequence information of the dnaK gene, C. lari forms two major distinct clusters consisting of UPTC and UN C. lari isolates, respectively, with UN C. lari being more closely related to other thermophilic campylobacters than to UPTC.

Molecular characterisation of urease genes from urease-positive thermophilic campylobacters (UPTC).

This study aims to clarify the molecular characteristics of the urease gene operon from urease-positive thermophilic campylobacters (UPTC) obtained from different sources and in various countries. Sequence heterogeneity was observed for the promoter structures at the -35-like region among the 12 isolates examined. The most probable TTG start codon was suggested for the ureB and ureH genes, and for the ureA, E, F and G genes, ATG was suggested among all the isolates examined. Overlap was detected between ureA and ureB and between ureB and ureE among all the isolates examined. UPTC is the first example of an overlap between the two structural genes ureA and ureB. When the completely sequenced open reading frames (ORFs) for ureE, ureF, ureG and ureH were identified, non-coding regions between ureE and ureF, ureF and ureG, and ureG and ureH were also demonstrated. All six start codons of the six urease genes were demonstrated to be preceded by Shine-Dalgarno sequences among all the isolates examined. The Cys-His sequence corresponding to urease active sites were aligned perfectly and fully conserved among the three UPTC isolates examined. A putative and intrinsic p-independent transcriptional terminator was identified to be identical among all the isolates examined. A partial and putative ORF of about 200 bp in length showing high sequence similarity to GTP cyclohydrolase I was observed downstream of ureH.

Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis.

Using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) of the 23S rRNA gene sequences of the genus Taylorella, none of the three representative T. equigenitalis strains NCTC11184(T), Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one approximately 70 bp IVS (TeIVS2) different from any IVSs found in T. asinigenitalis. The predicted secondary structure model of the IVSs contained stem and loop structures. The central region of the IVS-stem structure contains an identical double-stranded consensus 15-bp sequence. The purified RNA fraction from the three strains contained 16S and 4-5S RNA species but no 23S rRNA species. Thus, the primary 23S rRNA transcripts from the three strains would be cleaved into approximately 1.2- and 1.6-kb rRNA fragments and approximately 70-bp IVS. In addition, 16 other T. equigenitalis isolates were found to carry a similar 70-bp IVS in the central region and to produce fragmented 23S rRNA.

Cloning and structural analysis of the full-length cytolethal distending toxin (cdt) gene operon from Campylobacter lari.

Polymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdt gene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lari isolates using a new PCR primer pair constructed in silico. Three closely spaced and putative ORFs for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic p-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and cdtB and a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtA and cdtB and the non-coding region of six base pairs occurring between cdtB and cdtC. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six in cdtB and two in cdtC among the seven isolates (including C. lari RM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtB and mammalian DNase I were completely conserved in the cdtB gene locus in the 26 C. lari isolates, as well as in C. jejuni and C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC; n=10) using the primer pair.

Cloning, sequencing and molecular characterisation of a cryptic plasmid from a urease-positive thermophilic Campylobacter (UPTC) isolate.

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

Cloning, sequencing and characterization of a urease gene operon from urease-positive thermophilic Campylobacter (UPTC).

AIMS: To clone, sequence and characterize the genetic organization of urease genes within urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: An approx. 5.1-kbp region encoding a urease gene operon was identified, when recombinant plasmid DNAs from a genomic DNA library of a Japanese isolate (CF89-12) of UPTC were analysed. CONCLUSIONS: Six closely spaced and putative open reading frames (ORFs) for ureA, ureB, ureE, ureF, ureG and ureH were detected. ATG codons initiated each ORF of the UPTC urease operon except for ureB and ureH, which commenced with the most probable TTG codon. Overlaps were detected between ureA and ureB and also between ureB and ureE. Probable ribosome-binding sites and a putative rho-independent transcriptional termination region were identified. Two putative promoter structures, consisting of consensus sequences at the -35 like and -10 regions were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Construction of a neighbour-joining tree based on the nucleotide sequence data of urease genes indicated that UPTC formed a cluster with some Helicobacter organisms separate from the other urease-producing bacteria, suggesting a commonly shared ancestry between UPTC and Helicobacter urease genes.

Molecular characterization of the non-coding promoter and leader regions and full-length 16S ribosomal RNA (rRNA) gene of Taylorella asinigenitalis.

The 3,339 base pair (bp) sequences encoding a putative open reading frame (ORF), non-coding promoter and leader regions (approximately 320 bp), full-length 16S ribosomal RNA (rRNA) gene (approximate 1,540 bp) and part of the 16S-23S rDNA internal spacer region (ISR) were determined from genome DNA libraries of the Taylorella asinigenitalis (UK-1) isolate. The non-coding promoter and leader regions included antiterminators (boxB, boxA and boxC) immediately upstream of the 16S rRNA gene sequence. An approximately 680 bp region upstream of the non-coding promoter region appears to contain a putative ORF with high sequence similarity to GTP cyclohydrolase I. In addition, a typical order of intercistronic tRNA genes with the 48 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in a part of the 16S-23S rDNA ISR. The antiterminators of boxB and boxA were also identified in the ISR.A phylogenetic analysis based on the 16S rRNA gene sequence information clearly demonstrated that the five T. asinigenitalis isolates formed a cluster together with the three T. equigenitalis strains, more similar to Pelistega europaea than the other beta-Proteobacteria from the 13 species of 11 genera.

Flagellin gene structure of flaA and flaB and adjacent gene loci in urease-positive thermophilic Campylobacter (UPTC).

Two flagellin gene (flaA and flaB) sequences and the adjacent gene loci of urease-positive thermophilic Campylobacter (UPTC)(1) were examined. The flagellin gene sequences (1.7 kb) and adjacent gene loci of the two UPTC isolates (89049 and A3), obtained from animal hosts, were very similar to those of C. lari RM2100, C. jejuni, and C. coli. However, the structure and loci of the two flagellin genes (1.46-1.47 kb) and the adjacent gene loci of a UPTC strain obtained from the natural environment (NCTC12892) clearly differed from those of C. lari RM2100 and UPTC obtained from animal hosts. The two flagellin genes of UPTC 89049 and A3 were located between topA /CLA0518 and CLA0521, whereas those of NCTC12892 were located between topA and CLA0521. The sequences involved in regulation of flagellin expression, like sigma(28), sigma(54) and transcription termination signals, were conserved in all isolates. The characteristic direct repeat sequences containing a complete repeat unit of 5'-TCTTTAAAACAAC-3' were located in the intergenic regions between flaA and flaB in UPTC89049 and A3, but not in NCTC12892. The deduced amino acid sequence alignment revealed that the two flagellin genes in NCTC12892 had a deletion of the variable region of flagellin, which was reported previously to be modified by pseudaminic acid in C. jejuni and C. coli.Consequently, these results may possibly suggest that the length of the flagellin is related to pathogenicity and colonization of Campylobacter.

Demonstration of the shorter flagellin (flaA) gene of urease-positive thermophilic Campylobacter isolated from the natural environment in Northern Ireland.

The PCR amplicons (about 1450 bp in length) of flaA gene fragments of 11 isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from the natural environment not including wild birds in Northern Ireland were demonstrated to be shorter than those of C. jejuni 81116 and six isolates of C. jejuni and C. coli (about 1700 bp) isolated in Northern Ireland and Japan. When the nucleotide lengths of the possible open reading frame (ORF) of the flaA genes were determined, those from the 11 UPTC isolates were estimated to be 1464-1503 bp, and those from the six C. jejuni and C. coli isolates and C. jejuni 81116 strain to be 1716-1728 bp. Nucleotide sequence and deduced amino acid sequence alignments of the possible ORFs demonstrated that the ORFs from the 11 UPTC isolates lack about 80 amino acid residues, mainly from the approximate residue numbers 390-470 of the large variable region in the flaA protein of the seven isolates of C. jejuni and C. coli, and do not have any internal termination codons. High amino acid sequence similarity of both amino- and carboxy-termini of the ORFs of the flaA gene was demonstrated between the 11 isolates of UPTC and the 7 isolates of C. jejuni and C. coli. The 11 UPTC isolates examined were strongly suggested to possess a shorter flaA gene without any internal termination codons.

First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters (UPTC), and small restriction fragment sequencing.

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a p-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible p-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis.

BACKGROUND: At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. RESULTS: Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. CONCLUSION: High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.