A. Tazumi

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.

Identification and characterization of intervening sequences within 23S rRNA genes from more than 200 Campylobacter isolates from seven species including atypical campylobacters

BackgroundIdentification and characterization of intervening sequences (IVSs) within 23S rRNA genes from Campylobacter organisms including atypical campylobacters were carried out using two PCR primer pairs, designed to generate helix 25 and 45 regions.ResultsOnly C. sputorum biovar sputorum LMG7975 and fecalis LMG8531, LMG8534 and LMG6728 of a total of 204 Campylobacter isolates (n = 56 C. jejuni; n = 11 C. coli; n = 33 C. fetus; n = 43 C. upsaliensis; n = 30 C. hyointestinalis; n = 4 C. sputorum biovar sputorum; n = 5 C. sputorum biovar fecalis; n = 5 C. sputorum biovar paraureolyticus; n = 10 C. concisus; n = 7 C. curvus) were shown to carry IVSs in helix 25 region. C. sputorum biovar fecalis LMG8531 and LMG8534, interestingly, carried two different kinds of the 23S rRNA genes with and without the IVS, respectively. Consequently, in a total of 265 isolates of 269, including 65 C. lari isolates examined previously, the absence of IVSs was identified in the helix 25 region. In the helix 45 region, all the C. hyointestinalis, C. sputorum and C. concisus isolates were shown not to carry any IVSs. However, the 30 of 56 C. jejuni isolates (54%), 5 of 11 C. coli (45%), 25 of 33 C. fetus (76%), 30 of 43 C. upsaliensis (70%) and 6 of 7 C. curvus (90%) were shown to carry IVSs. In C. jejuni and C. upsaliensis isolates, two different kinds of the 23S rRNA genes were also identified to occur with and without IVSs in the helix 45 region, respectively.ConclusionsSecondary structure models were also constructed with all the IVSs identified in the present study. In the purified RNA fractions from the isolates which carried the 16S or 23S rRNA genes with the IVSs, no 16S or 23S rRNA was evident, respectively.

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.

Uneven distribution of the luxS gene within the genus Campylobacter.

Abstract Polymerase chain reaction (PCR) amplification was performed on 20 isolates of five Campylobacter species using a degenerate primer pair designed in silico to generate a product of the luxS gene or its homologue from Campylobacter organisms. Although the primer pair successfully amplified products of approximately 500 base pairs (bp) with the eight isolates of C. jejuni and C. coli and some of C. upsaliensis and C. fetus, it failed to amplify fragments with all four isolates of C. lari (two ureasenegative C. lari; two urease-positive thermophilic campylobacters). When Southern blot hybridisation analysis was carried using the mixed luxS gene fragments prepared from the C. jejuni, C. coli, C. upsaliensis and C. fetus strains as a probe, all C. jejuni, C. coli, C. upsaliensis and C. fetus isolates gave positive signals, but no positive signal was detected with any C. lari isolate. These results clearly indicate that C. jejuni, C. coli, C. upsaliensis and C. fetus carry the luxS gene or its homologue. However, no luxS gene or its homologue was identified to occur in the C. lari genome. Although autoinducer-2 assays were positive in C. jejuni, C. coli, C. upsaliensis and C. fetus isolates, it was negative with all the C. lari isolates examined. In addition, a biofilm formation assay demonstrated that biofilm formation in the C. lari species does not appear to correlate with the occurrence of the luxS gene because biofilm formation occurred among some isolates of C. lari.

Molecular and comparative analyses of the full-length cytolethal distending toxin (cdt) gene operon and its adjacent genetic loci from urease-positive thermophilic Campylobacter (UPTC) organisms.

Abstract Molecular and comparative analyses of the full-length cytolethal distending toxin (cdt) gene operon and its adjacent genetic loci (2.7–9.4 kilo base pairs in length) are carried out with 12 urease-positive thermophilic Campylobacter (UPTC) isolates using several polymerase chain reaction (PCR) primer pairs. Three putative open reading frames (ORFs) for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic ρ-independent transcription terminator were identified in all the operons of the 12 UPTC isolates examined. Although the number of amino acid residues slightly varied for the putative cdtA and cdtC ORFs, those for the cdtB were similar among all the UPTC isolates, as well as the six urease-negative (UN) C. lari examined previously. Regarding the cdt genes in UPTC CF89-12, each ORF commenced with an ATG start codon and terminated with a TAG stop codon for cdtA and cdtB and a TAA for cdtC. Start and stop codons of the three ORFs for the other 11 UPTC isolates were identical to those from the UPTC CF89-12 isolate except for the TTG start codon for cdtC in the two isolates (NCTC12892 and 12893) and the TGA stop codon for cdtA in five isolates (A1, A2, A3, 89049 and 92251). Two putative promoter structures, consisting of sequences at the –35-like (TTAATA) and –10-like (TATTAA) regions, as well as the start codon (ATG), were identified for the transcriptional promoter, immediately upstream of the cdtA gene in all the 12 isolates, Although the genetic heterogeneity of the cdtB gene locus occurred in all 28 C. lari isolates (n=16 UN C. lari; n=12 UPTC) examined, all nine amino acid-specific DNase residues were completely conserved in all their cdtB genes. Variable gene insertions with heterogeneous order and combinations occurred between cdtC and lpxB genes in the all UPTC organisms examined.Molecular and comparative analyses of the full-length cytolethal distending toxin (cdt) gene operon and its adjacent genetic loci (2.7-9.4 kilo base pairs in length) are carried out with 12 urease-positive thermophilic Campylobacter (UPTC) isolates using several polymerase chain reaction (PCR) primer pairs. Three putative open reading frames (ORFs) for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic ρ-independent transcription terminator were identified in all the operons of the 12 UPTC isolates examined. Although the number of amino acid residues slightly varied for the putative cdtA and cdtC ORFs, those for the cdtB were similar among all the UPTC isolates, as well as the six urease-negative (UN) C. lari examined previously. Regarding the cdt genes in UPTC CF89-12, each ORF commenced with an ATG start codon and terminated with a TAG stop codon for cdtA and cdtB and a TAA for cdtC. Start and stop codons of the three ORFs for the other 11 UPTC isolates were identical to those from the UPTC CF89-12 isolate except for the TTG start codon for cdtC in the two isolates (NCTC12892 and 12893) and the TGA stop codon for cdtA in five isolates (A1, A2, A3, 89049 and 92251). Two putative promoter structures, consisting of sequences at the -35-like (TTAATA) and -10-like (TATTAA) regions, as well as the start codon (ATG), were identified for the transcriptional promoter, immediately upstream of the cdtA gene in all the 12 isolates, Although the genetic heterogeneity of the cdtB gene locus occurred in all 28 C. lari isolates (n=16 UN C. lari; n=12 UPTC) examined, all nine amino acid-specific DNase residues were completely conserved in all their cdtB genes. Variable gene insertions with heterogeneous order and combinations occurred between cdtC and lpxB genes in the all UPTC organisms examined.

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.

Comparative analysis of Campylobacter lari cytolethal distending toxin (CDT) effect on HeLa cells

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81–176 and urease‐positive thermophilic Campylobacter (UPTC) CF89‐12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530T isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti‐recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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.

Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC).

Abstract This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF89- 12 showed enzyme activity over a broad pH range (pH 6–10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20–60°C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease α (A) and α (B) raised against Helicobacter pylori.