The Internal Transcribed Spacer Region, a New Tool for Use In

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The Internal Transcribed Spacer Region, a New Tool for Use in Species Differentiation and Delineation of Systematic Relationships within the Campylobacter Genusᰔ Si Ming Man,† Nadeem O. Kaakoush, Sophie Octavia, and Hazel Mitchell* School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia

Received 20 October 2009/Accepted 14 March 2010

The Campylobacter genus consists of a number of important human and animal pathogens. Although the 16S rRNA gene has been used extensively for detection and identiﬁcation of Campylobacter species, there is currently limited information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes. We examined the potential of the 23S rRNA gene and the ITS region to be used in species differentiation and delineation of systematic relationships for 30 taxa within the Campy- lobacter genus. The ITS region produced the highest mean pairwise percentage difference (35.94%) compared to the 16S (5.34%) and 23S (7.29%) rRNA genes. The discriminatory power for each region was further validated using Simpson’s index of diversity (D value). The D values were 0.968, 0.995, and 0.766 for the ITS region and the 23S and 16S rRNA genes, respectively. A closer examination of the ITS region revealed that Campylobacter concisus, Campylobacter showae, and Campylobacter fetus subsp. fetus harbored tRNA conﬁgura- tions not previously reported for other members of the Campylobacter genus. We also observed the presence of strain-dependent intervening sequences in the 23S rRNA genes. Neighbor-joining trees using the ITS region revealed that Campylobacter jejuni and Campylobacter coli strains clustered in subgroups, which was not observed in trees derived from the 16S or 23S rRNA gene. Of the three regions examined, the ITS region is by far the most cost-effective region for the differentiation and delineation of systematic relationships within the Campylobacter genus.

Members of the Campylobacter genus are Gram-negative, useful molecular marker for the study of phylogenetic relation- nutritionally fastidious, microaerophilic organisms that are spi- ships (28). However, this high sequence similarity observed ral, curved, or rod shaped and inhabit the gastrointestinal between members of the Campylobacter genus also makes it tracts of humans and animals. The first Campylobacter species difficult to differentiate between species such as C. jejuni and C. to be isolated was Campylobacter fetus (Vibrio fetus), isolated coli on the basis of the 16S rRNA gene (23, 43). In addition, from the uterine mucus of a sheep in 1906 (48). Since then, the this problem is further compounded by the remarkably similar Campylobacter genus has grown to comprise 17 formally phenotypic, host, and ecologic characteristics which many named species. The most recognized species within the Campy- Campylobacter species share. lobacter genus is Campylobacter jejuni, a gastrointestinal patho- Currently, there is relatively little sequence data available on gen and a leading bacterial cause of acute diarrhea and gas- the 23S rRNA gene and the internal transcribed spacer (ITS) troenteritis, accounting for 400 million cases in adults and region that lies between the 16S and 23S rRNA genes for children worldwide each year (2, 21, 44). Campylobacter coli members of the Campylobacter genus. Although the potential and a number of other non-jejuni Campylobacter species are of using the 23S rRNA gene and the ITS region for species also considered to be important human and animal pathogens differentiation and systematics has not been extensively inves- (1, 3, 14, 40, 56). tigated in the majority of Campylobacter species, previous stud- The 16S rRNA gene has been utilized extensively for rapid ies have examined the ability of these regions to differentiate a detection and identification of Campylobacter species (32, 36, small number of thermotolerant Campylobacter species, in- 37, 39). This is largely due to the fact that the 16S rRNA gene cluding C. jejuni, C. coli, Campylobacter lari, and Campy- is of considerable length (ϳ1,500 bp), and it is ubiquitous in lobacter upsaliensis (16, 18, 29). While the 23S rRNA genes in members of the Campylobacter genus and almost all bacteria a number of strains of C. jejuni and C. coli share a similar (9, 55). The fact that certain regions of the 16S rRNA gene are sequence identity (41), previous studies have reported the highly conserved, and that any changes in the sequence are presence of strain-specific intervening sequences (IVS) within therefore likely to be an accurate measure of time, makes it a the 23S rRNA gene of C. jejuni, C. coli, C. fetus, and C. upsaliensis (26, 53). Such an observation suggests that the 23S rRNA gene may be useful for differentiation at the strain level. * Corresponding author. Mailing address: School of Biotechnology While the presence of IVS elements does not appear to relate and Biomolecular Sciences, University of New South Wales, Sydney, to the pathogenicity in C. jejuni (31), IVS are known to lead to NSW 2052, Australia. Phone: 61 (2) 9385 2040. Fax: 61 (2) 9385 1591. rRNA fragmentation and have been hypothesized to be rem- E-mail: [email protected]. †Presentaddress:DepartmentofVeterinaryMedicine,Universityof nants of transposable elements (15, 35, 51, 53). Cambridge, Madingley Road, Cambridge CB3 0ES, United Kingdom. Within the 16S-ITS-23S operon, the ITS region in C. jejuni ᰔ Published ahead of print on 26 March 2010. subsp. jejuni, C. coli, and C. lari has been reported to be highly

3071 3072 MAN ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. Bacterial strains with a complete set of the 16S rRNA gene, 23S rRNA gene, and ITS regions sequenced in this study or obtained from GenBank

GenBank accession no. Species Strain Source of isolation 16S rRNA gene 23S rRNA gene ITS region C. coli ATCC 33559 Pig feces GQ167676 GQ167698 GQ167720 C. colia RM2228 Chicken carcass AAFL00000000 AAFL00000000 AAFL00000000 C. coli X7 Human feces GQ167671 GQ167695 GQ167716 C. coli X10 Human feces GQ167673 GQ167696 GQ167718 C. concisusa 13826 Human feces CP000792 CP000792 CP000792 C. concisus ATCC 51561 Human feces GQ167663 GQ167687 GQ167709 C. concisus ATCC 51562 Human diarrheic feces GQ167664 GQ167688 GQ167710 C. concisus UNSWCD Human colon GQ167662 GQ167686 GQ167708 C. curvusa 525.92 Human feces CP000767 CP000767 CP000767 C. fetus subsp. fetusa 82-40 Human blood CP000487 CP000487 CP000487 C. hominisa ATCC BAA-381 Human feces CP000776 CP000776 CP000776 C. hominis UNSWCD Human colon GQ167659 GQ167683 GQ167705 C. jejuni 100 Chicken carcass GQ167670 GQ167694 GQ167715 C. jejunia NCTC 11168 Human diarrheic feces AL111168 AL111168 AL111168 C. jejunia RM1221 Chicken skin CP000025 CP000025 CP000025 C. jejuni RP0001 Human colon GQ167656 GQ167680 GQ167702 C. jejuni INN-73-83 094400 Human diarrheic feces GQ167679 GQ167701 GQ167722 C. jejuni UNSW091300 Human feces GQ167677 GQ167699 GQ167721 C. jejuni subsp. doyleia 269.97/ATCC BAA-1458 Human feces CP000768 CP000768 CP000768 C. jejuni subsp. jejunia 81116 Human feces CP000814 CP000814 CP000814 C. jejuni subsp. jejunia 84-25 Human cerebrospinal ﬂuid AANT00000000 AANT00000000 AANT00000000 C. jejuni subsp. jejuni BABS091400 Human GQ167675 GQ167697 GQ167719 C. lari RM2100 Human diarrheic feces GQ167657 GQ167681 GQ167703 C. showae UNSWCD Human colon GQ167660 GQ167684 GQ167706 C. upsaliensis RM3195 Human feces GQ167658 GQ167682 GQ167704 B. ureolyticusb UNSWC Human feces GQ167661 GQ167685 GQ167707 B. ureolyticus UNSWE Human feces GQ167667 GQ167691 GQ167712 B. ureolyticus UNSWJ Human feces GQ167668 GQ167692 GQ167713 B. ureolyticus UNSWM Human colon GQ167669 GQ167693 GQ167714 B. ureolyticus UNSWR Human feces GQ167665 GQ167689 GQ167711 A. butzleria RM4018 Human feces CP000361 CP000361 CP000361

a The 16S and 23S rRNA genes and ITS regions were extracted from the whole genome available from GenBank. b Bacteroides ureolyticus is a taxonomically misclassiﬁed species belonging to the Campylobacter genus (54).

variable in size and/or sequence composition and contains dif- CO2 microaerophilic conditions generated by the Campylobacter system ferent types of sequences encoding tRNA molecules (8, 29). BR0056A (Oxoid Limited, Hampshire, United Kingdom). Bacteria were har- The diversity of the ITS region observed previously in these vested and washed once using phosphate-buffered saline (PBS) prior to DNA extraction. Additional near-complete 16S and 23S rRNA genes and ITS regions Campylobacter species suggests that this region may be useful from a range of Campylobacter species, not available from our collection, were for differentiation and identification of other nonthermotoler- obtained from GenBank (http://www.ncbi.nlm.nih.gov/). ant Campylobacter species. Indeed, the ITS region has increas- DNA extraction and PCR amplification of the 16S rRNA gene, 23S rRNA gene, ingly been used for differentiation between bacterial species or and ITS region. Bacterial DNA was extracted using the Qiagen Puregene core kit strains, including Escherichia coli strains (20), Mycobacterium A (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Campylobacter species DNA was subjected to PCR analysis using combinations spp. (47), cyanobacteria (4, 46), and acetic acid bacteria (50), of 18 PCR primers to amplify the 16S rRNA gene, ITS region, and 23S rRNA which cannot be easily differentiated using the 16S rRNA gene. gene (Table 2). The target position of each primer is illustrated in Fig. 1. One Furthermore, the versatility of the ITS region as a molecular pair of primers was used in a single PCR. PCR analysis was performed in a 25-␮l tool has also been exploited in environmental microbiology in reaction mixture consisting of 10 pmol of each primer pair (Sigma-Aldrich, the study of marine microbial diversity (6, 7, 19, 49). Sydney, Australia), 1ϫ PCR buffer (Fisher Biotech, Subiaco, Australia), 200 nM each deoxynucleotide triphosphate (Fisher Biotech), 1.5 mM MgCl2 (Fisher The aim of this study was therefore to examine the ability of Biotech), 0.7 U Taq polymerase (Fisher Biotech), and 20 ng DNA. The thermal the 16S rRNA gene, 23S rRNA gene, and ITS region to dif- cycling conditions for the PCR were as follows: 94°C for 5 min, 40 cycles of 94°C ferentiate and delineate systematic relationships between 30 for 20 s, 52°C for 20 s, and 72°C for 2 min, followed by 72°C for 7 min. Five members of the Campylobacter genus. microliters of the PCR product was subjected to gel electrophoresis (1.5% agarose), stained with 1ϫ GelRed nucleic acid gel staining solution (Biotium, Hayward, CA) for 10 min, and visualized under UV transillumination. MATERIALS AND METHODS Sequence identification. PCR products were sequenced using BigDye Termi- Bacterial cultivation. The Campylobacter taxa used in this study were obtained nator chemistry (Applied Biosystems, Foster City, CA) on an ABI 3730 capillary from our own laboratory, the University of New South Wales Culture Collection, DNA sequencer (Applied Biosystems). Prior to the sequencing reaction, PCR the American Type Culture Collection (ATCC), or the National Collection of products were purified using the QIAquick PCR purification kit (Qiagen) ac- Type Cultures (NCTC), as shown in Table 1. Arcobacter butzleri RM4018, a cording to the manufacturer’s instructions. Sequencing of both the 5Ј and 3Ј ends species from a genus closely related to the Campylobacter genus, was used in this of the amplicons was performed, using 1 ␮l ABI Prism BigDye Terminator study for comparative analysis. All bacteria were grown on horse blood agar version 3.1 (Applied Biosystems), 10 pmol/␮l of the required primer, 50 to 200 supplemented with 5% defibrinated horse blood for 2 to 4 days at 37°C under 5% ng DNA, and Milli-Q water (Millipore, Bedford, MA) to make up the final VOL. 76, 2010 ITS REGION OF CAMPYLOBACTER SPECIES 3073

TABLE 2. PCR primers used in the amplification of the 16S rRNA from an additional 10 members of the Campylobacter genus gene, 23S rRNA gene, and ITS region of Campylobacter species were obtained from GenBank (30 sequences). Sequences of Primer Sequence Reference each region from 30 Campylobacter isolates were used to de- termine inter- and intraspecies sequence variability. The mean F27 5Ј-AGAGTTTGATCCTGGCTCAG-3Ј 33 1494R 5Ј-TACGGCTACCTTGTTACGAC-3Ј 33 pairwise percentage difference between all Campylobacter spe- 1494R(RC) 5Ј-GTCGTAACAAGGTAGCCGTA-3Ј This study cies when using the ITS sequence was 35.94%, which was RC1494M 5Ј-GTCGTAACAAGGTAACCGT-3Ј This study significantly higher than the 5.34% and 7.29% obtained when M83 5Ј-KTTCGCTCGCCRCTAC-3Ј 11 using 16S and 23S rRNA gene sequences, respectively (P Ͻ M83M 5Ј-TACGGGACTATCACCCTCTA-3Ј This study O68 5Ј-AGGCGATGAAGGACGTA-3Ј 11 0.0001; paired t test). The discriminatory power for each indi- O68M 5Ј-AGGCGATGAAAGACGTG-3Ј This study vidual region was calculated using Simpson’s index of diversity M85 5Ј-AGTRAGCTRTTACGC-3Ј 11 (D value) (25), where a D value of 1 denotes the greatest ability M85M 5Ј-ACCAGTGAGCTATTACGC-3Ј This study of a region in discriminating different isolates. The 23S rRNA 43a 5Ј-GGATGTTGGCTTAGAAGCAG-3Ј 52 69ar 5Ј-CTTAGGACCGTTATAGTTAC-3Ј 52 gene had the highest D value (0.995), followed closely by the 16S1F 5Ј-GACACACGTGCTACAATG-3Ј This study ITS region (D value ϭ 0.968), while the 16S rRNA gene had 16S2R 5Ј-TGACCTCACCCTTATCAG-3Ј This study the lowest D value (0.766). 23S1F 5Ј-GATGACTTGTGGATAGGG-3Ј This study Comparison of the sequences from the ITS region between 23S2R 5Ј-CTGTGTCGGTTTACGGTA-3Ј This study M94 5Ј-AAACCGWCACAGGTRG-3Ј 11 species showed that Campylobacter curvus 525.92 and Campy- M89 5Ј-CTTAGATGCYTTCAGC-3Ј 11 lobacter hominis ATCC BAA381 were the most variable (70.21% pairwise difference). The ITS region was able to differentiate between the two C. hominis strains (25.85% difference), which were found to be identical when using the 16S volume of 20 ␮l. The sequencing program consisted of 96°C for 1 min and 30 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. rRNA gene. However, the ITS region was identical in all of the Analyses of sequence variability, diversity, and systematic relationships be- four C. coli strains, and therefore it could not be used for tween members of the Campylobacter genus using the 16S rRNA gene, 23S rRNA differentiation of the strains within this species. gene, and ITS region. Analyses were performed using programs available from The highest pairwise percentage difference between strains the Australian National Genomic Information Service (ANGIS) at the Univer- sity of Sydney. PILEUP from the GCG package (12) and Multicomp (45) were of the same species was between C. hominis ATCC BAA-381 used for multiple sequence alignment and comparison. The average pairwise and C. hominis UNSWCD (25.85%). The intraspecies pairwise percent difference was calculated with Multicomp using a method described by differences using the ITS region for other Campylobacter spe- Nei and Miller (42). The ability of an individual region to discriminate isolates C. coli Campylobacter was further determined using Simpson’s index of diversity (D value) performed cies were as follows: , 0%, four strains; as previously described (25) using the following formula: concisus, 1.19 to 5.49%, four strains; C. jejuni (including subspecies), 0 to 12.84%, 10 strains; and Bacteroides ureolyticus, S 1 0.7 to 7.69%, five strains. The ITS region produced the highest D ϭ 1 Ϫ n ͑n ͒ N͑N Ϫ 1͒͸ j j Ϫ 1 pairwise percentage difference between strains of C. jejuni, C. j ϭ 1 concisus, C. hominis, and B. ureolyticus compared to the 16S where N is the total number of isolates analyzed, S is the total number of rRNA gene and the 23S rRNA gene. Campylobacter sequence types obtained, and nj is the number of isolates belong- Comparison of the 16S rRNA gene sequences between spe- ing to the jth type. Phylip was used to generate neighbor-joining trees and bootstrap values (17). Secondary structures for Campylobacter 23S rRNA se- cies showed that C. curvus 525.92 and B. ureolyticus UNSWE quences were predicted using the GeneBee program (5) available from Moscow were the most variable (11.74% difference). The 16S rRNA State University (http://www.genebee.msu.su/services/rna2_reduced.html). The gene sequence was unable to differentiate the majority of C. Aragorn program was used for the identification of different types of tRNA jejuni and C. coli strains from each other. For example, the 16S molecules, the number of bases, and % GC content within the ITS region (34). rRNA gene sequence of C. coli X7 and C. coli X10 was identical to four other strains of C. jejuni. RESULTS The highest pairwise percentage difference between strains Inter- and intraspecies sequence variability of the 16S rRNA of the same species was 1.43% (between C. coli ATCC 33559 gene, 23S rRNA gene, and ITS region between members of the and C. coli RM2228/C. coli X7/C. coli X10). The intraspecies Campylobacter genus. The 16S rRNA gene, 23S rRNA gene, differences using the 16S rRNA gene sequences for all other and ITS region of 20 members of the Campylobacter genus Campylobacter species were as follows: C. coli, 0 to 1.43%, four were generated by using PCR (60 sequences), and sequences strains; C. concisus, 0.15 to 0.45%, four strains; C. hominis, 0%,

FIG. 1. Schematic representation of the 18 forward (3) and reverse (4) primers and their target positions within the 16S-ITS-23S rRNA operon. 3074 MAN ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 3. Type, nucleotide position, no. of bases, and % GC content of tRNA molecules identiﬁed within the ITS regions

ITS First ITS tRNA Second ITS tRNA Overall Species Strain length Nucleotide No. of Nucleotide No. of % GC Type % GC Type % GC (bp) position bases position bases C. coli ATCC 33559 876 30.6 tRNAAla(TGC) 103–178 76 60.5 tRNAIle(GAT) 187–263 77 53.2 C. coli RM2228 980 32.6 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. coli X7 875 30.6 tRNAAla(TGC) 103–178 76 60.5 tRNAIle(GAT) 186–262 77 53.2 C. coli X10 875 30.6 tRNAAla(TGC) 103–178 76 60.5 tRNAIle(GAT) 186–262 77 53.2 C. concisus 13826 698 34.5 tRNAIle(GAT) 97–173 77 53.2 tRNAAla(TGC) 186–261 76 60.5 C. concisus ATCC 51561 592 33.3 tRNAIle(GAT) 97–173 77 53.2 tRNAAla(TGC) 186–261 76 60.5 C. concisus ATCC 51562 592 32.8 tRNAIle(GAT) 97–173 77 53.2 tRNAAla(TGC) 186–261 76 60.5 C. concisus UNSWCD 591 33.8 tRNAIle(GAT) 97–173 77 53.2 tRNAAla(TGC) 186–261 76 60.5 C. curvus 525.92 1,129 33.4 tRNAAla(TGC) 297–372 76 60.5 tRNAIle(GAT) 661–737 77 53.2 C. fetus subsp. fetus 82-40 917 33.3 tRNAAla(TGC) 145–220 76 60.5 —b —b —b —b C. hominis ATCC BAA-381a 1,646 22.7 tRNAAla(TGC) 303–378 76 60.5 tRNAIle(GAT) 464–540 77 54.5 C. hominis UNSWCDa 1,177 24 tRNAAla(TGC) 308–383 76 60.5 tRNAIle(GAT) 469–545 77 54.5 C. jejuni 100 806 28 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni NCTC 11168 806 28.4 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni RM1221 801 28.6 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni RP0001 799 28 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni INN-73-83 094400 714 29.4 tRNAAla(TGC) 103–178 76 60.5 tRNAIle(GAT) 187–263 77 53.2 C. jejuni UNSW091300 806 27.7 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni subsp. doylei 269.97 828 28.3 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni subsp. jejuni 81116 805 27.5 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni subsp. jejuni 84-25 806 28 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. jejuni subsp. jejuni BABS091400 806 28 tRNAAla(TGC) 105–180 76 60.5 tRNAIle(GAT) 189–265 77 53.2 C. lari RM2100 837 29.5 tRNAAla(TGC) 253–328 76 60.5 tRNAIle(GAT) 335–411 77 53.2 C. showae UNSWCD 671 33.4 tRNAIle(GAT) 122–198 77 53.2 tRNAAla(TGC) 211–286 76 60.5 C. upsaliensis RM3195a 893 33.9 tRNAAla(TGC) 114–189 76 60.5 tRNAIle(GAT) 197–273 77 53.2 B. ureolyticus UNSWE 1,021 25.5 tRNAAla(TGC) 149–224 76 60.5 tRNAIle(GAT) 228–304 77 53.2 B. ureolyticus UNSWC 1,023 25.2 tRNAAla(TGC) 155–230 76 60.5 tRNAIle(GAT) 234–310 77 53.2 B. ureolyticus UNSWJ 916 27.4 tRNAAla(TGC) 149–224 76 60.5 tRNAIle(GAT) 228–304 77 53.2 B. ureolyticus UNSWM 1,057 24.5 tRNAAla(TGC) 149–224 76 60.5 tRNAIle(GAT) 228–304 77 53.2 B. ureolyticus UNSWR 1,051 25 tRNAAla(TGC) 149–224 76 60.5 tRNAIle(GAT) 228–304 77 53.2 A. butzleri RM4018 704 26.6 tRNAIle(GAT) 107–183 77 55.8 tRNAAla(TGC) 238–313 76 60.5

a Campylobacter species with the 16S rRNA gene and 23S rRNA gene in separate locations. The region identified to be immediately downstream of the 16S rRNA gene and upstream of the next open reading frame was used as the ITS region. b Absence of a second tRNA molecule. two strains; C. jejuni (including subspecies), 0 to 0.23%, 10 from C. concisus UNSWCD. In addition, all C. concisus strains strains; and B. ureolyticus, 0.08 to 0.0.38%, five strains. harbored ITS regions that were relatively short in length (591 Comparison of the 23S rRNA gene sequences between spe- to 698 bp) but higher in % GC contents (32.8 to 34.5%) in cies showed that C. curvus 525.92 and C. jejuni subsp. doylei comparison with other Campylobacter species. The average % 269.97 were the most variable (13.82% difference). The 23S GC content of the Campylobacter ITS region was 29.3%. C. rRNA gene was unable to distinguish two strains of C. coli concisus 13826 had the highest % GC content in the ITS region (strains X7 and X10) and two strains of C. jejuni (strains 100 at 34.5%, while C. hominis ATCC BAA-381 had the lowest % and BABS091400). GC content at 22.7%. The variability in length and % GC The highest percentage of pairwise difference between content was also evident between strains of a specific Campy- strains of the same species was observed between C. jejuni lobacter species. The lengths of the ITS region for 10 C. jejuni INN-73-83 094400 and C. jejuni subsp. doylei 269.97 (1.55% strains ranged from 714 bp (C. jejuni toxigenic strain Mexico difference). Examination of the 23S rRNA gene showed that INN-73-83 094400) to 828 bp (C. jejuni subsp. doylei 269.97), the intraspecies differences for the remaining Campylobacter and the % GC content ranged from 27.5% (C. jejuni subsp. species were as follows: C. coli, 0 to 0.47%, four strains; C. jejuni 81116) to 29.4% (C. jejuni toxigenic strain Mexico INN- concisus, 0.12 to 2.51%, four strains; C. hominis, 0.51%, two 73-83 094400). strains; C. jejuni (including subspecies), 0 to 1.55%, 10 strains; All Campylobacter species examined in this study had a 16S- and B. ureolyticus, 0.04 to 0.28%, five strains. ITS-23S operon configuration except for C. hominis and C. Variability of the ITS region in members of the Campy- upsaliensis.The16Sand23SrRNAgenesofthesetwospecies lobacter genus. The ITS region is a region which lies between were not arranged in an operon. The 16S-ITS-23S operon was the 16S rRNA gene and the 23S rRNA gene. The ITS regions determined by PCR amplification, which generates a visible prod- of 30 members of the Campylobacter genus were highly vari- uct using primers that target a region spanning the 16S-ITS or able in length and % GC content. The average length of the ITS-23S region. No PCR amplicon was observed when the ITS Campylobacter ITS region was 880 bp. The longest ITS region region of C. hominis and C. upsaliensis was amplified by PCR was 1,646 bp, which was observed in C. hominis ATCC BAA- analysis using primers 1494R(RC) or RC1494M (forward primers 381 (Table 3). The shortest ITS region was 591 bp in length which anneal to the 3Ј end of the 16S rRNA gene) and M83 or VOL. 76, 2010 ITS REGION OF CAMPYLOBACTER SPECIES 3075

M83M (reverse primers which anneal to the 5Ј end of the 23S to that observed in the 16S rRNA gene. The second cluster rRNA gene), whereas PCR amplicons were visible for other consisted of C. concisus, C. showae, and C. curvus. Cluster III Campylobacter species tested in this study. Examination of whole consisted of C. fetus subsp. fetus and two C. hominis strains. genomes from fully sequenced C. hominis ATCC BAA-381 The clustering of C. fetus subsp. fetus differed in the 23S rRNA (GenBank accession no. CP000776) and C. upsaliensis RM3195 gene tree compared with the 16S rRNA gene tree in which C. (GenBank accession no. AAFJ00000000) confirmed that these fetus subsp. fetus clustered with C. jejuni, C. coli, C. lari, and C. species do not have the 16S-ITS-23S operon. The region identi- upsaliensis. B. ureolyticus strains formed a distinct cluster (clus- fied to be immediately downstream of the 16S rRNA gene and ter IV), similar to that of the tree derived from the 16S rRNA upstream of the next open reading frame was used as the ITS gene. region for C. hominis and C. upsaliensis,giventhatinthisregion For the NJ gene tree based on Campylobacter sequences tRNA coding genes were identified in a configuration similar to derived from the ITS region, a markedly different topology was that of other Campylobacter species. observed, characterized by the presence of three clusters (Fig. Configurations of tRNAs in the ITS region. The following 2C). All B. ureolyticus strains formed a discrete cluster (cluster three tRNA configurations were observed in 30 Campylobacter I), consistent with observations in the trees derived from the taxa: (i) tRNAAla(TGC) and tRNAIle(GAT), (ii) tRNAIle(GAT) 16S and 23S rRNA genes. Cluster II included all strains of C. and tRNAAla(TGC), and (iii) tRNAAla(TGC) (Table 3). C. fetus jejuni, C. coli, and C. lari, which was congruent with the clus- subsp. fetus 82-40 was the only Campylobacter species with tering of thermotolerant Campylobacter species observed when tRNA configuration 3, a configuration with a single tRNA- using the 16S and 23S rRNA genes. A closer inspection of the coding gene. The tRNA-coding gene for tRNAAla(TGC) was tree revealed that the ITS region was able to further divide C. identified at position 145 to 220 within the ITS region of C. jejuni and C. coli strains into subclusters, with the exception of fetus subsp. fetus 82-40, but no tRNAIle(GAT) was identified. All one strain of C. jejuni (INN-73-83 094400) which subclustered Campylobacter species except for C. concisus and Campy- with the C. coli strains. Cluster III contained C. concisus and C. lobacter showae had tRNA configuration 1, characterized by showae. However, C. curvus did not cluster with C. concisus the presence of tRNAAla(TGC) followed by tRNAIle(GAT) (the and C. showae, as was observed using the 16S and 23S rRNA most prevalent configuration). C. concisus, C. showae, and A. gene data. Interestingly, cluster III was more closely related to butzleri had tRNA configuration 2, which contained a reversed A. butzleri than to the remaining Campylobacter taxa. The re- order of the tRNA-coding genes compared to configuration 1. latedness of C. concisus and C. showae to A. butzleri was also All of the identified tRNAAla(TGC) molecules consisted of 76 reflected by their unique tRNA configuration (configuration 2, bases, and all of the tRNAIle(GAT) molecules consisted of 77 16S-tRNAIle(GAT)-tRNAAla(TGC)-23S), unlike most other bases (Table 3). The % GC content in all tRNAAla(TGC) was Campylobacter species (configuration 1, 16S-tRNAAla(TGC)- 60.5%, which was highly conserved. In contrast, the % GC tRNAIle(GAT)-23S), as highlighted earlier. C. fetus subsp. fetus, content in tRNAIle(GAT) was more variable, with all Campy- C. curvus, C. hominis, and C. upsaliensis were very divergent lobacter species having a GC content of 53.2% except C. and did not cluster with other Campylobacter species. hominis ATCC BAA-381 and C. hominis UNSWCD strains, Using gene sequences from the 16S and 23S rRNA genes which had a GC content of 54.5%. A. butzleri RM4018 had and the ITS region, we generated an NJ tree using all three atRNAIle(GAT) GC content of 55.8%, which was greater than all regions (combined tree). Five major clusters were generated of the Campylobacter species examined in this study. (Fig. 2D). Cluster I consisted of C. jejuni strains only. Cluster Systematic analysis of the 16S rRNA gene, 23S rRNA gene, II consisted of all C. coli strains and C. jejuni INN-73-83 and ITS region using the neighbor-joining method. Sequences 094400. The combined tree was the only tree that was able to of the 16S rRNA gene, 23S rRNA gene, and ITS region were clearly group C. jejuni and C. coli strains into distinct clusters aligned to generate neighbor-joining (NJ) trees. A. butzleri with the exception of C. jejuni INN-73-83 094400. Clusters III RM4018 (accession no. CP000361) was used as an outgroup. In and IV were comprised of C. hominis and B. ureolyticus, re- the NJ tree derived from the 16S rRNA gene sequences, there spectively, showing grouping congruency with trees derived were four major clusters (Fig. 2A). Cluster I consisted of very from three individual regions. C. concisus, C. showae, and C. closely related C. coli and C. jejuni strains, C. lari, C. fetus curvus formed one group (cluster V), which is comparable to subsp. fetus, and C. upsaliensis. Thermotolerant Campylobacter the clustering using individual regions. C. fetus subsp. fetus, C. species which have the ability to grow at 42°C were found only lari, and C. upsaliensis were very divergent and did not cluster in cluster I (C. jejuni, C. coli, C. lari, and C. upsaliensis). C. fetus with other Campylobacter species. Thermotolerant Campy- subsp. fetus, which is not considered a thermotolerant Campy- lobacter species did not form one distinct cluster but were lobacter species, also clustered in cluster I. However, some closely related to each other, with C. jejuni and C. coli taxa strains of C. fetus subsp. fetus have been reported to grow at positioned in clusters I and II, respectively, and C. lari and C. 42°C (13, 24). Cluster II contained all B. ureolyticus strains upsaliensis located in close proximity to clusters I and II. only. Cluster III included both C. hominis strains. Cluster IV Presence of IVS in the 16S and 23S rRNA genes contributed comprised of C. concisus, C. showae, and C. curvus. to the variability of members of the Campylobacter genus. The In the NJ tree derived from the 23S rRNA gene sequences, 16S and 23S rRNA gene sequences were aligned against their members of the Campylobacter genus were divided into four corresponding genes in E. coli strain K-12 (accession no. major clusters (Fig. 2B), similar to the tree derived from the NC000913). Intervening sequences (IVS) were present in the 16S rRNA gene data. The first cluster contained C. jejuni, C. 16S rRNA gene of one of the 30 Campylobacter taxa (3%). An coli, C. upsaliensis, and C. lari. All thermotolerant Campy- IVS of approximately 200 bp in size was found in C. curvus lobacter species were grouped in this cluster in a fashion similar 525.92. The IVS was located at position 220 with respect to the 3076 MAN ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 2. Neighbor-joining (NJ) trees based on Campylobacter sequences derived from the 16S rRNA gene (A), 23S rRNA gene (B), ITS region (C), and all three regions combined (D). Each taxon is labeled by species and strain number, and in brackets is the original source of isolation. Bootstrap values, if greater than 50%, are presented at nodes of the tree.

16S rRNA gene in E. coli K-12. The presence of IVS was also There were six different IVS types, ranging from 37 bp (C. observed in the 23S rRNA gene sequences of 12 of the 30 jejuni UNSW091300 and C. jejuni subsp. jejuni 81116) to 240 Campylobacter taxa (40%). While there was only one IVS iden- bp (C. curvus 525.92). The most frequently detected IVS was tified in the 23S rRNA gene of each taxon, the IVS differed in 143 bp in length and in position 1023 (with respect to E. coli length, position, and type (Table 4). Not all of the strains K-12), and this type of IVS was found in five Campylobacter within a specific species had identical IVS lengths, positions, taxa, including three C. coli strains (RM2228, X7, and X10) and sequences. For example, three types of IVS were identified and two C. jejuni strains (INN-73-83 094400 and RM1221). in C. jejuni. Two Campylobacter taxa (C. jejuni subsp. doylei 269.97 and C. VOL. 76, 2010 ITS REGION OF CAMPYLOBACTER SPECIES 3077

FIG. 2—Continued. upsaliensis RM3195) harbored a 172-bp IVS at position 1024. ing 11 strains which did not have IVS included all ﬁve B. C. jejuni UNSW091300 and C. jejuni subsp. jejuni 81116 har- ureolyticus strains (UNSWC, UNSWE, UNSWJ, UNSWM, bored a 37-bp IVS at position 1204. C. curvus 525.92, C. fetus UNSWR), all four C. concisus strains (13826, ATCC 51561, subsp. fetus 82-40, and C. hominis UNSWCD all harbored a ATCC 51562, UNSWCD), C. lari,andC. showae. C. curvus unique type of IVS with variable lengths and positions. 525.92 was the only Campylobacter species examined to have con- The presence of IVS in the Campylobacter genus appeared tained IVS elements in both the 16S and 23S rRNA gene. to be strain dependent. Of the four strains of C. coli examined, Spatial distribution of the IVS in relation to the secondary three contained IVS of the same sequence at the same loca- structure of 23S rRNA molecules. The secondary structure of tion. C. coli strain ATCC 33559 was the only C. coli strain the 23S rRNA molecules harboring an IVS from all Campy- without an IVS in its 23S rRNA gene. Furthermore, IVS were lobacter taxa was predicted. As shown in Fig. 3A using C. present in 5 of 10 strains of C. jejuni, including three IVS of curvus 525.92 as a representative, the IVS was located at one different sequence composition at two distinct positions. The extremity and was not a part of the central structure of the ﬁve C. jejuni strains that did not contain an IVS were C. predicted 23S rRNA molecule. Removal of the IVS from the jejuni 100, 84-25, BABS 091400, NCTC 11168, and RP0001. sequence of the 23S rRNA gene resulted in no major change to C. hominis strain UNSWCD also contained an IVS, whereas the secondary structure of the 23S rRNA molecule (Fig. 3B). none was found in C. hominis ATCC BAA381. The remain- The predicted secondary structures of two fragmented 23S

TABLE 4. Length, position, and type of IVS in Campylobacter species and size of fragmented 23S rRNA molecules following excision of the IVS

Size (bp) of fragmented IVS length Nucleotide Species Strain IVS typeb 23S rRNA (bp) positiona 12 C. coli RM2228 143 1,023 1 1,185 1,721 C. coli X7 143 1,023 1 1,183 1,472 C. coli X10 143 1,023 1 1,183 1,472 C. curvus 525.92 240 1,200 2 1,178 1,696 C. fetus subsp. fetus 82-40 119 1,198 3 1,079 1,567 C. hominis UNSWCD 105 1,196 4 1,099 1,515 C. jejuni INN-73-83 094400 143 1,023 1 1,183 1,476 C. jejuni RM1221 143 1,023 1 1,185 1,721 C. jejuni UNSW091300 37 1,204 5 1,183 1,472 C. jejuni subsp. doylei 269.97 172 1,204 6 1,180 1,699 C. jejuni subsp. jejuni 81116 37 1,204 5 1,183 1,721 C. upsaliensis RM3195 172 1,204 6 1,182 1,535

a Positions based on E. coli K-12 numbering system. b Six types of unique IVS were identiﬁed within the Campylobacter genus. 3078 MAN ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. The representative secondary structures of the Campylobacter 23S rRNA molecule using C. curvus 525.92. Shown is the 23S rRNA secondary structure with (A) and without (B) the 240-bp IVS. The spatial distribution of IVS within the secondary structure of the 23S rRNA is indicated in a box (A). The predicted secondary structures of two resultant fragmented 23S rRNA molecules are shown in panels C and D. The locations of the neighboring regions are represented by triangles. rRNA molecules obtained after excision of the IVS are shown M83M. All Campylobacter species investigated possessed iden- in Fig. 3C and D. tical 16S and 23S rRNA genes despite multiple copies of rrn Multiple 16S-ITS-23S operons in members of the Campy- operons. lobacter genus. Examination of available whole genomes of 10 Campylobacter isolates (C. coli RM2228, C. concisus 13826, C. DISCUSSION curvus 525.92, C. fetus subsp. fetus 82-40, C. hominis ATCC BAA-381, C. jejuni NCTC 11168, C. jejuni RM1221, C. jejuni The Campylobacter genus consists of a number of important subsp. jejuni 81116, C. lari RM2100, and C. upsaliensis pathogens in human and veterinary medicine. In this study, we RM3195) showed that only three members of the Campy- investigated the characteristics of the 16S rRNA gene, 23S lobacter genus harbored multiple rrn operons that consisted of rRNA gene, and ITS region for 30 taxa within the Campy- variable ITS regions. Two sequence compositions of the ITS lobacter genus and their potential to be used in species differ- region were observed in C. concisus 13826, C. fetus subsp. fetus entiation and delineation of systematic relationships. Similar to 82-40, and C. coli RM2228. To address whether the variability the closely related members of the Helicobacter genus, IVS can of the ITS regions affected the systematic relationship, the two be found in both the 16S and 23S rRNA genes (11, 35). In this versions of the ITS region from C. concisus 13826, C. fetus study, we identified the presence of IVS elements in the 23S subsp. fetus 82-40, and C. coli RM2228 were included in an NJ rRNA gene of 12 of the 30 taxa within the Campylobacter tree generated from the ITS region. The two different ITS genus. We have shown for the first time that C. curvus and C. sequences of C. concisus 13826 and C. fetus subsp. fetus 82-40 hominis also contain an IVS element in their 23S rRNA genes. remained in their respective clusters and were not in conflict In addition, IVS of various lengths within the 23S rRNA gene (data not shown). In C. coli RM2228, both ITS sequences were were identified in different strains of C. jejuni, C. coli, C. fetus, clustered in the C. jejuni/C. coli cluster. One ITS sequence of and C. upsaliensis, which is in agreement with previously re- C. coli RM2228 clustered with the C. coli subcluster within the ported studies (26, 31, 51). The strain-specific appearance of C. jejuni/C. coli cluster, whereas the second ITS sequence clus- IVS within the 23S rRNA gene suggests that this gene would tered closer to the C. jejuni subcluster. No other C. coli strains be useful for differentiation between strains that contain highly examined in this study appear to have variable ITS sequences similar or identical 16S rRNA gene sequences. Our study re- following sequencing of the 5Ј and 3Ј ends of the ITS region vealed that of the Campylobacter species examined, none had using the ITS primer pair RC1494M/1494R(RC) and M83/ more than one IVS within the 23S rRNA gene. In contrast, VOL. 76, 2010 ITS REGION OF CAMPYLOBACTER SPECIES 3079 members of the phylogenetically related Helicobacter genus Another major taxonomic problem in the Campylobacter have been shown to contain one or more IVS within the 23S genus is the taxonomic position of B. ureolyticus, which is rRNA gene, in four distinct positions (11). Interestingly, the currently ambiguous. Vandamme et al. (54) conducted a presence of IVS is known to lead to rRNA fragmentation polyphasic taxonomic study of B. ureolyticus and found that this induced by posttranscriptional excision, which mediates the species has a quinone content, DNA base ratio, and pheno- removal of IVS from within the rRNA molecule of bacteria, typic characteristics similar to those of other Campylobacter including Campylobacter species and members of the Alpha- species, differing only in its fatty acid composition and ability to proteobacteria (15, 31, 51, 53). Ribosomal RNAs without IVS digest casein and gelatin (54). remain intact after transcription. The evolutionary advantage On the basis of the 16S and 23S rRNA genes and the ITS and function of these IVS elements is, however, still unclear. region, our genotypic analyses of five B. ureolyticus strains Thus far, no relationship between C. jejuni strains containing indicated that they were most closely related to C. hominis. an IVS and their pathogenicity has been observed (31). This This bacterium is morphologically similar to B. ureolyticus, suggests that IVS may not have an essential function and that both of which are nonmotile, rod-shaped organisms that ex- they may be remnants of transposable elements, which have hibit a swarming characteristic on horse blood agar, possibly been previously inserted into and subsequently excised from mediated by the use of pili. In addition, on the basis of the 16S the 23S rRNA genes (31, 35). rRNA gene and the ITS region, we have shown that C. jejuni The ITS region found between the 16S and 23S rRNA genes and C. coli were more closely related to B. ureolyticus than they has been previously investigated only in a small group of were to C. concisus, C. curvus, and C. showae. Given this new Campylobacter species consisting of C. jejuni, C. coli, and C. evidence of a close systematic grouping of B. ureolyticus with lari, but not in any other members of the Campylobacter genus. other members of the Campylobacter genus on the basis of These studies have shown that all the Campylobacter species regions other than the 16S rRNA gene, there is an increasing investigated contain a 5Ј-16S-tRNAAla-tRNAIle-23S-3Ј tRNA prospect of classifying B. ureolyticus as a formal member of the configuration (8, 30). Examination of other members of the Campylobacter genus. Campylobacter genus in our study revealed two other possible Comparative sequence analyses of the 16S and 23S rRNA tRNA configurations. The most prevalent configuration was genes and the ITS region of members of the Campylobacter found to be the 5Ј-16S-tRNAAla-tRNAIle-23S-3Ј configuration, genus revealed that the most discriminatory region for species which was observed in all Campylobacter species except C. and strain differentiation was the ITS region. While the 23S concisus, C. showae, and C. fetus subsp. fetus. In C. concisus and rRNA gene has the highest D value, it has a relatively low C. showae, we observed an inverted tRNA configuration not mean pairwise difference. In contrast, the ITS region has a previously reported in members of the Campylobacter genus significantly higher mean pairwise difference and a D value (5Ј-16S-tRNAIle-tRNAAla-23S-3Ј). This inverted tRNA config- similar to that of the 23S rRNA gene. Although it is clear that uration has also been shown in Bacillus subtilis (38) and cya- when the 16S and 23S rRNA genes and the ITS region were nobacteria (27). The second configuration not previously re- combined, a resultant NJ tree elicited taxonomic relationships ported was found in C. fetus subsp. fetus, which had only one of the highest resolution between members of the Campy- tRNA-coding gene (tRNAAla), a characteristic that is found lobacter genus. However, one of the major limitations involved primarily in Gram-positive bacteria (22). in examining all three regions is the time, effort, and cost The differentiation of C. jejuni and C. coli continues to be a required to amplify, sequence, and assemble a region of 5,500 significant taxonomic problem, as both species share remark- nucleotides in length. Indeed, the entire process required 14 ably similar phenotypic, genotypic, host, and ecologic charac- different PCR and sequencing primers. Of the three regions teristics and are virtually indistinguishable using the 16S or 23S examined, the ITS region is by far the best region for bacterial rRNA gene, as shown in this study. Currently, the most reliable identification, differentiation, and systematic analysis, as it is method to distinguish C. jejuni and C. coli is believed to be the the shortest (ϳ1,000 bp) and is highly discriminatory. In con- presence of the hippuricase gene in C. jejuni but absent in C. trast to the four and eight different PCR and sequencing prim- coli (36). Interestingly, delineation of the systematic relation- ers required to obtain the near-complete 16S rRNA gene ships between members of the Campylobacter genus showed (1,500 bp) and 23S rRNA gene (2,500 to 3,000 bp), only one that when the 16S and 23S rRNA genes and the ITS region pair of primers is required for the amplification and sequenc- were combined, we were able to clearly differentiate strains of ing of the complete ITS region. 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