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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 urease-negative 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.

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 CF8912 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 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

The primer pair (C412F/C1228R) constructed previously for the polymerase chain reaction (PCR) identification of the genus Campylobacter using an approximate 800 base pair (bp) 16S rRNA gene target segment proved to be useful for the identification of a total of 49 Campylobacter lari isolates including urease-positive thermophilic Campylobacter (UPTC) organisms (n=25). When the primer pair (CLF/R) developed previously for the PCR identification of C. lari species using an approximate 250 bp glyA segment was employed, 27 C. lari isolates, including all the UPTC isolates, were identified to be PCR-negative (55%). Therefore, this PCR procedure developed for the molecular identification of C. lari was shown to be unreliable for C. lari identification. Nucleotide sequencing analysis clarified the reason(s) why PCR-negative examples occurred in many C. lari isolates, including UPTC isolates. The primer pair target sequences in the C. lari-specific PCR-negative isolates apparently varied at the 3' end region, as compared with C. lari-specific PCR-positive isolates. Thus, the multiplex PCR assay developed previously was shown to be unreliable for the molecular identification of C. lari subspecies organisms.

A polymerase chain reaction (PCR) method was carried out on 21 isolates of atypical Campylobacter sputorum (n=14) and C. curvus (n=7) using a primer pair to amplify the helix 11 region within 16S ribosomal RNA (rRNA) gene sequences. Following sequencing and alignment analysis, 14 C. sputorum (100%) and six C. curvus (86%) isolates were shown to carry intervening sequences (IVSs) in this region. Interestingly, the nucleotide sequences of all the IVSs were identical among the 14 C. sputorum isolates (n=5 C. sputorum biovar [bv] paraureolyticus; n=5 by fecalis; n=4 by sputorum). In addition, two different nucleotide lengths and sequences of IVSs were identified among the six C. curvus isolates. On the first prediction of the secondary structure model of the IVSs in 16S rRNA genes, stem and loop structures were identified. In the purified RNA fractions from the 20 Campylobacter isolates carrying IVSs, no 16S rRNA was evident. Instead, other smaller RNA fragments were identified. Thus, the primary 16S rRNA transcripts may have been fragmented in the 20 isolates. This is the first demonstration of atypical C. sputorum and C. curvus isolates carrying IVSs in the helix 11 region in 16S rRNA genes.

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 rho-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.

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.

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.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

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.

(2009). Absence of intervening sequences (IVSs) in helix 11 region within 16S rRNA genes among more than 240 isolates of the seven Campylobacter species. British Journal of Biomedical Science: Vol. 66, No. 2, pp. 103-106.

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.

(2010). Do equine strains of Pseudomonas aeruginosa carry the Liverpool epidemic strain markers relevant to patients with cystic fibrosis? British Journal of Biomedical Science: Vol. 67, No. 1, pp. 30-31.