T. Sekizuka

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

BackgroundAt 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.ResultsClarification 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.ConclusionHigh sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

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).

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

Abstract 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 ρ-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.

Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF) and its adjacent genetic loci within Campylobacter lari.

BackgroundThe combined sequences encoding a partial and putative rpsI open reading frame (ORF), non-coding (NC) region, a putative ORF for the Campylobacter adhesin to fibronectin-like protein (cadF), a putative Cla_0387 ORF, NC region and a partial and putative Cla_0388 ORF, were identified in 16 Campylobacter lari isolates, using two novel degenerate primer pairs. Probable consensus sequence at the -35 and -10 regions were identified in all C. lari isolates, as a promoter.ResultsThus, cadF (-like) gene is highly conserved among C. lari organisms. Transcription of the cadF (-like) 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 (-like) ORFs from the C. lari isolates.ConclusionThe putative cadF (-like) ORFs from all C. lari isolates were nine amino acid larger than those from C. jejuni, and showed amino acid residues 137 -140 of FALG (50% identity), 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 (-like) gene sequence information formed a major cluster consisting of C. lari isolates, separating from the other three thermophilic campylobacters.

Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis

An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other β-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the β-Proteobacteria.

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

The PCR amplicons (about 1450 bp in length) offlaA gene fragments of 11 isolates of urease-positive thermophilicCampylobacter (UPTC) isolated from the natural environment not including wild birds in Northern Ireland were demonstrated to be shorter than those ofC. jejuni 81116 and six isolates ofC. jejuni andC. coli (about 1700 bp) isolated in Northern Ireland and Japan. When the nucleotide lengths of the possible open reading frame (ORF) of theflaA genes were determined, those from the 11 UPTC isolates were estimated to be 1464–1503 bp, and those from the sixC. jejuni andC. coli isolates andC. 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 theflaA protein of the seven isolates ofC. jejuni andC. coli, and do not have any internal termination codons. High amino acid sequence similarity of both amino- and carboxy-termini of the ORFs of theflaA gene was demonstrated between the 11 isolates of UPTC and the 7 isolates ofC. jejuni andC. coli. The 11 UPTC isolates examined were strongly suggested to possess a shorterflaA gene without any internal termination codons.

flaA‐like sequences containing internal termination codons (TAG) in urease‐positive thermophilic Campylobacter isolated in Japan

Aims: To demonstrate two flaA‐like sequences containing two internal termination codons (TAG) in two Japanese strains of urease‐positive thermophilic Campylobacter (UPTC). 
Methods and Results: A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx. 1450 bp for two Japanese strains of UPTC, CF89‐12 and CF89‐14. 
After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. 
Conclusions: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. 
Significance and Impact of the Study: Surprisingly, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.

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 NCTC11184T, Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one ≈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 ≈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.

Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari

coli, are curved Gram-negative bacteria that are the recognised cause of acute bacterial diarrhoea around the world. C. lari is a relatively recently discovered thermophilic Campylobacter species 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. An atypical group of isolates of urease-positive thermophilic campylobacters (UPTC) was first isolated from the natural environment in England in 1985. 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 Ireland, in The Netherlands and in Japan. The possible association of UPTC with human disease remains unclear. Two representative taxa, namely ureasenegative (UN) C. lari and UPTC, occur within the species of C. lari. Although several Campylobacter species’ cytotoxins have been identified, only the cytolethal distending toxin (CDT) has been characterised in detail. The cdt genes of C. jejuni have been cloned and characterised by Pickett et al. However, in relation to the cdt genes, no reports have yet appeared for C. lari. Therefore, the aim of the present study is to clone, sequence and analyse the cdtB gene of C. lari isolates and compare the sequences obtained with those of other thermophilic campylobacters. Twenty-four isolates of C. lari (UN C. lari [n=16] and UPTC [n=8]) were used in the present study (Table 1), together with three reference strains (JCM2530T, NCTC12892 and NCTC12893). The test organisms were isolated from different sources in several countries. The organisms were cultured on blood agar containing defibrinated horse blood (Nippon Bio-Test, Tokyo, Japan) and supplemented with campylobacter-selective medium (Nissui, Tokyo, Japan), under microaerophilic conditions at 37 ̊C for two days. Template DNA was prepared by boiling in water at 95 ̊C for five minutes. The PCR mixture contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 400 μmol each dNTP, 1 μmol each primer, and 1 unit of Thermus aquaticus (Taq) DNA polymerase (Takara Bio, Shiga, Japan). A schematic representation of the cdtB gene and its genetic loci for C. lari RM2100 (GenBank Accession No. AAFK00000000) including the locations of a primer pair for the cdtB (JCB common up and JCB common down [Asakura primer]) employed in the present study for PCR amplification is shown in Figure 1. This primer pair was designed to generate a product of approximately 700 bp (equivalent to a 90% segment of the cdtB structural gene of C. lari RM2100, AAFK00000000) of the cdtB gene with C. jejuni, C. coli and C. fetus isolates. The polymerase chain reaction (PCR) was performed in 50-μL reaction volumes, for 30 cycles at 94 ̊C for 30 sec, 55 ̊C for 30 sec, 72 ̊C for 45 sec, followed by a final extension of 72 ̊C for 5 min. Amplified PCR products were separated by 1.0% (w/v) agarose gel electrophoresis in 0.5x TBE at 100 V and detected by ethidium bromide staining. PCR products amplified by the constructed primer pair for the partial cdtB gene were purified using a QIA quick PCR purification kit (Qiagen, CA, USA) and inserted in the pGEM-T vector using the pGEM-T Easy Vector System (Promega, Tokyo, Japan). Sequencing of the cloned cdtB gene fragment was performed (Hitachi DNA autosequencers SQ-5500L and SQ5500EL) after a dideoxy nucleotide sequencing reaction, using a Thermo Sequenase premixed cycle sequencing kit (Amersham Pharmacia Biotech, Tokyo, Japan). Sequence analysis of the PCR amplicons was carried out using the GENETYX-MAC (version 9) computer software. Genetic heterogeneity of the cytolethal distending toxin B (cdtB) gene locus among isolates of Campylobacter lari

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

Abstract 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.