Takuya Nakajima

Molecular identification of an arsenic four-gene operon in Campylobacter lari

An arsenic (ars) four-gene operon, containing genes encoding a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC) and an arsenical-resistance membrane transporter (Acr3) was first identified in urease-positive thermophilic Campylobacter (UPTC) isolate, CF89-12. UPTC CF89-12 and some other Campylobacter lari isolates contained their ars four-genes, similarly, differing from that in the reference C. lari RM2100 strain. Two putative promoters and a putative terminator were identified for the operon in UPTC CF89-12. In vivo transcription of the operon was confirmed in the UPTC cells. PCR experiments using two primer pairs designed in silico to amplify two arsR and arsC-acr3 segments, respectively, generated two amplicons, approximately 200 and 350 base pairs, with all 31 of 31 and 19 of 31 C. lari isolates (nu2009=u200917 for UPTC; nu2009=u200914 for UN C. lari), respectively. An inverted repeat forming a dyad structure, a potential binding site for a transcriptional repressor, was identified in the promoter region. Within the deduced 61 amino acids sequence of the putative arsR open reading frame from the UPTC CF89-12, a metal binding box and a DNA-binding helix-turn-helix motif were identified. The UPTC CF89-12 and some other UPTC isolates isolated from natural environment were resistant to arsenate.

Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837–844 base pair (bp) [nxa0=xa0six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530T, RM2100, 176, 293, 299 and 448] and short length ISRs of 679–725xa0bp [nxa0=xa0six for UN C.lari: nxa0=xa014 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156xa0bp sequence from the nucleotide positions 122–277xa0bp in long length ISRs for UN C. lari JCM2530T. The 156xa0bp sequences shared 94.9–96.8xa0% sequence similarity among six isolates. Surprisingly, atypical tRNAAla gene segment (5′ end 35xa0bp), which was extremely truncated, occurred within the 156xa0bp sequences in the long length ISRs, as an unexpected tRNAAla pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5′-16S rDNA-tRNAAla-tRNAIle-23S rDNA-3′ occurred in all the C. lari isolates examined.

Correlations between 23S rRNA genes and erythromycin resistance in Campylobacter jejuni

There has been no description of the prevalence of intervening sequences (IVSs) within the 23S rRNA genes in clinical Campylobacter jejuni isolated from humans, and, moreover, no studies have yet appeared describing the correlation between nucleotide sequences of 23S rRNA genes and erythromycin (Ery) resistance in human isolates. Minimum inhibitory concentration of 49 human clinical C. jejuni isolates isolated in Asia, Europe, and North America were determined, and 8 isolates were resistant to Ery (≥ 8xa0μg/ml; 16%). Following sequencing and alignment analyses, no correlations between the nucleotide sequences of the V domain and the IVSs within 23S rRNA gene sequences and Ery resistance occurred in the human clinical C. jejuni isolates examined. In addition, no point mutations occurred at any expected and putative positions in the V domain with the isolates. In addition, no correlations were seen between the occurrence of the IVSs and random amplified polymorphic DNA subtypes of the C. jejuni isolates. Therefore, ABC efflux pump and other resistance mechanisms(s) may be involved in the resistance to Ery in the human clinical C. jejuni isolates examined in the present study.

Molecular cloning and characterisation of the methionine sulphoxide reductase A (msrA) gene locus in Campylobacter lari organisms.

Abstract The methionine sulphoxide reductase A (msrA) gene and its adjacent genetic loci from urease-negative (UN) Campylobacter lari RM2100 and urease-positive thermophilic Campylobacter (UPTC) CF89–12 strains appear to be composed of a msrA structure gene (507 base pairs [bp]) and another five-gene cluster (approximately 6300 bp) in the same strand and direction. A primer pair (F1/R4-msrA) for polymerase chain reaction (PCR) amplification was designed to generate a product of approximately 900 bp of the msrA gene, including its adjacent genetic loci for the thermophilic Campylobacter organisms and generate an amplicon with 16 C. lari isolates (n=4 for UN C. lari; n=12 for UPTC). Following direct nucleotide sequencing, sequence analysis and nucleotide sequence alignment analysis, the putative full-length msrA gene from the 16 C. lari isolates showed high nucleotide sequence similarities (91.8–100%) to each other and relatively low similarity (69.3–71.8%) to three reference C. jejuni and C. coli strains. In addition, the msrA gene was transcribed in both the UPTC CF89–12 and NCTC12893 cells using reverse transcription PCR. An immunoreactively positive signal was identified in the UPTC CF89–12 and NCTC12893 cells with anti-UPTC MsrA synthetic peptide antibodies.

Molecular identification and characterization of type III restriction-modification (R-M) gene cluster in Campylobacter lari

Although the human clinical strain of Campylobacter lari (RM2100) has been shown not to carry any type III restriction-modification (R-M) systems, an R-M genes cluster was identified downstream of the full-length cytolethal distending toxin gene operon in the urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain. Two possible open reading frames (ORFs) for restriction endonuclease and methyltransferase were predicted to encode peptides of 947 and 613 amino acid residues with calculated molecular weights of 111 and 70.8 kDa, respectively. Two putative promoters consisting of the consensus sequences and two probable ribosome binding sites for the two ORFs were also identified. Reverse transcription PCR identified co-transcription of the R-M genes in the cells. The existence of an S-adenosyl methionine-binding motif in the N-terminal conserved region of the possible ORF for the M gene, and seven conserved helicase motifs in the R gene were also identified. PCR and Southern blot hybridization analyses for type III R-M enzyme genes with some of the C. lari isolates including UPTC gave positive signals. UPTC isolates were shown to carry type III R-M enzyme genes, with a relatively high frequency.

Molecular analysis of the tlyA gene in Campylobacter lari

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000xa0base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000xa0bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720xa0bp with a calculated molecular mass of approximately 26.7xa0kDa. Using a primer pair designed in silico, a total of approximately 1.1xa0kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [nu2009=u200913 for UPTC; nu2009=u20094 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90xa0bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

Molecular analysis of superoxide dismutase in Campylobacter lari

The superoxide dismutase (SOD) gene clusters, sodB and sodC, and their adjacent genetic loci from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain were analyzed molecularly, and compared with those of thermophilic campylobacters. The UPTC CF89-12 strain carried sodB [structural gene 654 base pairs (bp)] and sodC (540xa0bp) genes, as did the Campylobacter lari RM2100 reference strain. However, the other three thermophilic Campylobacter jejuni, C. coli and C. upsaliensis reference strains carried only a single sodB gene, and no sodC. Although sodB and sodC in the UPTC strain shared relatively high nucleotide sequence similarities (92.9xa0% and 91.7xa0%, respectively) with the corresponding genes in the C. lari RM2100 strain, the sodB gene in the UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni NCTC11168 (80.8xa0% and 81.7xa0%), C. coli RM2228 (82.0xa0% and 83.1xa0%) and C. upsaliensis RM3195 (75.9xa0% and 77.0xa0%), respectively. All PCR amplifications of sodB and sodC gene segments with 28 C. lari isolates, including 14 UPTC isolates, gave positive results. C. lari organisms were shown to carry both the sodB and sodC genes with extremely high frequency. More high-SOD activity was seen with the C. lari isolates (nu2009=u20099), including UPTC, than was seen with the other three thermophilic Campylobacter and Helicobacter pylori organisms.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in Campylobacter lari

PCR amplifications using primers for the clustered regularly interspaced short palindromic repeats (CRISPRs)-associated gene 1 (cas1), cas2, putative (p)-cas and CRISPRs genes generated cas1, cas2, p-cas and CRISPRs genes segments with 9–28 of 28 urease-positive thermophilic Campylobacter (UPTC) isolates, respectively. The p-cas and CRISPRs genes segments were amplified with 10 of 11 and 0 of 11 urease-negative (UN) Campylobacterlari isolates, respectively. When the nucleotide sequences of the CRISPRs consensus sequence repeats of each 33–37 base pairs from the 18 Campylobacterjejuni isolates were aligned, as well as from the four C. jejuni reference and UPTC CF89-12 strains, the repeats were identified as being almost identical. Although a total of all 18 C. jejuni isolates examined gave PCR-positive signals for the CRISPRs genes, it was, interestingly, suggested that many numbers of C. lari and C. jejuni isolates may possibly carry cas but not CRISPRs genes within their CRISPRs loci. In addition, PCR amplification by using a novel primer pair of f-ClCRISPR-ladder and ClCRISPRs-R, which were novel to this study, with the UPTC CF89-12 strain was shown to be useful for the detection of the putative CRISPRs separated by the non-repetitive unique spacer regions, with the electrophoretic ladder DNA profile following 5.0xa0% polyacrylamide gel electrophoresis. Secondary structure models of the CRISPRs repeats were predicted with UPTC CF89-12 and two C. jejuni strains.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000xa0base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32xa0bp; 5′-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3′). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the −35 and −10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170xa0bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Absence of intervening sequences and point mutations in the V domain within 23S rRNA in Campylobacter lari isolates.

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [nu2009=u200919 for urease-positive thermophilic Campylobacter (UPTC); nu2009=u200916 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8xa0μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16xa0μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.