1 Institute of Medical Microbiology and Immunology, University of Aarhus, Denmark
2 Department of Community Oral Health and Pediatric Dentistry, University of Aarhus, Denmark
Correspondence
Knud Poulsen
kp{at}microbiology.au.dk
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ABSTRACT |
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INTRODUCTION |
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Members of the JP2 clone are serotype b and characterized by a 530 bp deletion in the promoter region of the ltx operon which encodes the leukotoxin (Brogan et al., 1994). This deletion results in enhanced production of the toxin. In addition, strains of the JP2 clone are distinguished from other types of A. actinomycetemcomitans by a deleterious nonsense mutation in the haemoglobin-binding-protein gene hgpA, which implies that they are unable to utilize human haemoglobin as a source of iron (Hayashida et al., 2002
).
The highly leukotoxic strains of A. actinomycetemcomitans are identical in multilocus enzyme electrophoresis (MLEE) analysis and they have the same DNA fingerprint when using the restriction enzyme MspI (Haubek et al., 1996, 1997
). Thus, these strains constitute a clone. However, RFLP analyses of EcoRI-digested genomic DNA using rrn genes encoding rRNA as well as the IS150-like insertion sequence as hybridization probes have revealed differences among strains within the clone (Haubek et al., 1996
, 1997
; Hayashida et al., 2000
). Here, we show that these differences are caused by homologous intragenomic recombination involving the six copies of rrn genes and the seven IS150-like elements, resulting in translocations or large inversions within the genome.
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METHODS |
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Multilocus sequence typing.
Internal fragments of approximately 500 bp of six housekeeping genes were amplified by PCR and sequenced. The genes were: pgi, encoding glucose-6-phosphate isomerase; recA, encoding RecA protein; adk, encoding adenylate kinase; frdB, encoding fumarate reductase; atpG, encoding the gamma subunit of ATP synthase F1; and mdh, encoding malate dehydrogenase. The primers designed from the genome sequence of strain HK1651 are listed in Table 1. For amplification in a volume of 25 µl we used approximately 1 ng whole-cell DNA as template, 10 pmol of each primer, and Hot Master Mix (Eppendorf). The temperature profile for the PCR was: denaturation at 94 °C for 5 min followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min, followed by a final extension at 72 °C for 8 min. The PCR products were purified using Wizard Minicolumns (Promega). For DNA sequencing we used the same primers as for the PCR together with the Thermo Sequenase dye terminator cycle sequencing kit (Amersham Life Science). The resulting products were analysed with an Applied Biosystem PRISM 377 automated sequencer (Perkin-Elmer Applied Biosystem). The regions sequenced in the ORF of the six genes were: 296822 in pgi, 55549 in recA, 32590 in adk, 215724 in frdB, 23522 in atpG and 182621 in mdh. For sequence analyses we used the program CLUSTALX (Thompson et al., 1997
).
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Southern blot analysis.
Approximately 5 µg of whole-cell DNA was digested with EcoRI and electrophoresed as described above. The DNA fragments in the gel were transferred and fixed onto a nylon membrane and hybridized as previously described, with a final medium-stringency wash in 1x SET (0·15 M NaCl, 0·5 mM EDTA, 20 mM Tris/HCl, pH 7·0), 0·1 % SDS and 0·1 % sodium pyrophosphate at 60 °C (Hayashida et al., 2000). The probes used were prepared by PCR using DNA from strain HK1651 as template. The primers used for amplification of the first third of the 16S rRNA gene (corresponding to positions 24534 in the Escherichia coli 16S rRNA gene) were 5'-TATTACCGCGGCTGCTGGCA-3' and 5'-TCAGATTGAACGCTGGCGGC-3', and for the IS150-like element probe we used 5'-ATTTCCGCCTGTAATTCGGCAATCG-3' combined with 5'-ACATCGCATATTGCCCCGAATGTG-3', which amplify a 0·4 kb fragment of an IS150-like element identified by searching the A. actinomycetemcomitans HK1651 genome sequence, as described by Hayashida et al. (2000)
. The PCR products were purified from the agarose gel after electrophoresis using the Qiaex II Gel Extraction kit (Qiagen) and labelled with [32P]dCTP using a Random Primed Labelling kit (Roche).
PCR typing.
The six rrn gene sequences and flanking regions from the genome sequence of strain HK1651 were aligned, and unique primer sequences in the flanking regions were identified. The primers used are shown in Table 1. Since the expected size of the amplicons was approximately 7 kb, we used the Expand Long Template PCR System as recommended by the supplier (Roche). The thermocycling parameters were as follows: denaturation at 94 °C for 3 min followed by 25 cycles at 94 °C for 15 s, 60 °C for 30 s and 68 °C for 8 min, and ending with a final extension at 68 °C for 8 min. A long extension time was used in the PCR because of the large size of the amplicons and in order to avoid PCR jumping or bridging. The resulting DNA fragments were analysed by 1 % agarose gel electrophoresis.
Similarly, the sequences of the seven IS150-like elements and flanking regions in the HK1651 genome were aligned, and flanking primers unique for the amplification of each element were synthesized. The seven pairs of primers are listed in Table 1. The expected size of the PCR fragments was approximately 1·7 kb, and for the amplifications we used Hot Master Mix (Eppendorf) with the following thermocycling program: denaturation at 94 °C for 5 min and 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min, followed by a final extension at 72 °C for 8 min. The PCR products were analysed by 1 % agarose gel electrophoresis.
I-CeuI and XhoI DNA fingerprinting.
Samples were digested with I-CeuI (New England BioLabs) or XhoI (Roche) and analysed by PFGE. I-CeuI specifically cleaves a sequence in the 23S rRNA gene and XhoI was selected because it has 20 recognition sites in the A. actinomycetemcomitans strain HK1651 genome sequence and was therefore anticipated to be suitable for genomic fingerprinting using PFGE. The procedures were as described by Hansen et al. (2004), with modifications. Most strains were of the adherent rough phenotype. These strains aggregate when grown in liquid cultures, and to facilitate preparation of the agarose plugs they were grown for 2 days on chocolate agar plates. Five millilitres of Tris/EDTA (TE) buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7·4) was added to the plate and the bacteria were washed off the plate using a spatula. After centrifugation, the cells were suspended in 0·5 ml TE buffer 10 : 100 (10 mM Tris, 100 mM EDTA, pH 8) and treated with 100 µg proteinase K for 1 h at 37 °C to resolve aggregation of the bacteria mediated by proteins. The cell suspension was mixed 1 : 1 with 2 % low-melting-point agarose to prepare the agarose plugs. The proteinase K was inactivated by incubation of the plugs in 1·5 ml of 1·43 mM PMSF in TE buffer twice for 30 min, followed by washing once in water and four times for 30 min in 2 ml TE buffer. The plugs were treated with lysozyme (1 mg lysozyme ml1, 1 % sarcosyl in TE buffer 10 : 100) for 2 h at 37 °C, followed by digestion with proteinase K overnight at 50 °C, as described by Hansen et al. (2004)
. After washing, the plugs were equilibrated in the I-CeuI or XhoI buffer supplied with the enzymes and digested overnight in 100 µl containing 30 U of the restriction enzyme, followed by twice adding an additional 30 U of enzyme and incubating for 20 h each time. We found that it was quite difficult to obtain a complete digest of the genomic DNA in the plugs, and therefore the restriction enzymes were added three consecutive times. The DNA fragments were separated by PFGE in a 1 % agarose gel in Tris/borate/EDTA buffer at 14 °C, using the GenePath System (Bio-Rad) and the preprogrammed run condition PSU.
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RESULTS |
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DISCUSSION |
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All five strains analysed differed in the combinations of primers that resulted in amplification of the six rrn gene copies. Thus, although belonging to the same clone, they are all of different genome type. The rrn gene operons contain EcoRI sites. Therefore, homologous recombination between different rrn genes outside the EcoRI fragment containing the 16S rRNA sequences used as probe does not influence the EcoRI RFLP typing using this probe. Consequently, strains identical in the EcoRI RFLP typing (Fig. 1b, lanes 1, 2 and 3) may have different genome types reflected in distinct combinations of primers resulting in successful amplification of the six rrn operons (Table 2
). It is not possible to deduce the rrn gene arrangement in the common ancestor from the data presented in Table 2
. Theoretically, the rrn skeleton in strain HK1519 may have arisen from strain HK1651 by a recombination between rrn2 and rrn4 resulting in an inversion. The differences between strains HK1651 and HK1200 could be explained by a recombination between rrn2 and rrn3 in the HK1651 genome (Fig. 2a
), followed by a recombination between rrn1 and the novel chimeric rrn2/rrn3 gene at the position of rrn3, or by excision by recombination between rrn1 and rrn2 followed by transposition into rrn3 by homologous recombination. Creation of the rRNA gene arrangement in strains HK921 and HK1199 from either of the others would involve more recombination events.
The PCR analysis revealed limited variation in sequences flanking the IS150-like elements (Table 3). An intrinsic characteristic of insertion sequences is the ability to shift position in the genome. For all strains, the Southern blot analysis indicated the presence of seven copies of this element (Fig. 1c
), and the PCR analysis showed that seven combinations of flanking primers resulted in the amplification of a strong band (Table 3
). Thus, there is no evidence of excision, amplification or transposition of this insertion sequence, and thus integrations of this element within the chromosome seem to be stable. The differences in combinations of flanking primers resulting in amplification of a DNA fragment suggest that a recombination between IS150-2 and IS150-4 has taken place in strain HK1651 compared to the other strains. Strains HK1519, HK921, HK1200 and HK1199 showed an identical pattern of sequences flanking the IS150-like elements, though in the EcoRI Southern blot analysis, a single band differed between HK1199 and the other strains (Fig. 1c
). Theoretically, this could be explained by a recombination involving the 3' end of rrn3, since in the HK1651 genome, this rRNA gene is located very close to IS150-3, and there is no EcoRI restriction site in the intervening region (Fig. 2a
). Thus, a recombination between rrn3 and rrn2 or rrn1 might influence the IS150-like EcoRI RFLP pattern without changing the sequences right next to the IS150-like element. Alternatively, a point mutation may have created/deleted a flanking EcoRI site and thereby changed the size of the EcoRI fragment that hybridizes with the fragment.
The HK1651 strain used for the PCR analyses was transferred to a new plate every week for more than one year in the laboratory, and yet the arrangement of the rrn genes and IS150-like sequences was the same as in the parental strain used for genome sequencing. This indicates that successful intragenomic recombination between these multi-copy sequences is a rare event when the bacterium is grown in vitro. However, we cannot exclude that such changes in the genome may be more frequent in vivo, because they might provide a selective advantage in the environment of the human host.
The inversions and translocations were reflected in differences in XhoI DNA fingerprinting using PFGE. There are 20 recognition sites for XhoI in the HK1651 genome sequence, and only a part of the resulting fragments is resolved by PFGE; that is, the smaller fragments migrate together (Fig. 4). This may explain why strains HK921 and HK1199 show the same XhoI fingerprint, though they differ in the RFLP analyses. Strains HK921 and HK1519 were identical in the RFLP analyses using 16S rRNA gene sequences and the IS150-like element as probes, and yet they have different XhoI fingerprints. As described above, this is most likely due to homologous recombination between rrn genes in the region outside the EcoRI fragment that hybridized with the 16S rRNA probe used in the Southern blot analysis. XhoI DNA fingerprinting of 20 strains isolated within one year from adolescents all living in Morocco revealed eight different types. This basic variation in a temporally and geographically restricted population of the JP2 clone of A. actinomycetemcomitans provides a means to study local epidemiology, such as transmission of this virulent bacterium. However, care should be taken in drawing conclusions relating to the long-term evolution of the JP2 clone from the results of XhoI DNA fingerprinting, because homologous recombinations are reversible events.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 May 2005;
revised 1 July 2005;
accepted 11 July 2005.
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