Intragenomic recombination in the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans

Kirsten T. Eriksen1, Dorte Haubek2 and Knud Poulsen1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans is strongly associated with aggressive periodontitis in adolescents of African descent. DNA fingerprinting using the frequently cutting restriction enzyme MspI and multilocus sequence typing (MLST) showed that five strains of this clone were genetically virtually identical, although ribotyping of the six rrn genes and EcoRI RFLP analysis of the seven IS150-like elements revealed differences. PCR analyses demonstrated that these multi-copy sequences are subject to intragenomic homologous recombination, resulting in translocations or large inversions. The genome rearrangements were reflected in differences among 25 strains representing the JP2 clone in DNA fingerprinting using the rare-cutting restriction enzyme XhoI and resolved by PFGE. XhoI DNA fingerprinting provides a tool for studying local epidemiology, including transmission of this particularly pathogenic clone of A. actinomycetemcomitans.


Abbreviations: MLST, multilocus sequence typing


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Actinobacillus actinomycetemcomitans is a Gram-negative rod that is often present in subgingival dental plaque of humans. Members of the highly leukotoxic JP2 clone constitute a particularly pathogenic subpopulation of A. actinomycetemcomitans. Strain JP2, isolated from an 8 year old African-American boy with prepubertal periodontitis, has served as the prototype of this clone (Tsai et al., 1984). The presence of the clone is strongly associated with periodontal attachment loss among adolescents, and healthy children harbouring this bacterium are more likely to convert to aggressive periodontitis (Bueno et al., 1998; Haubek et al., 2001, 2004). Notably, strains of the JP2 clone of A. actinomycetemcomitans have almost exclusively been isolated from subjects of North and West African descent (Contreras et al., 2000; Haraszthy et al., 2000; Haubek et al., 1996, 1997). The reason for this tropism for certain ethnic populations is not yet known.

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
A total of 25 isolates of A. actinomycetemcomitans were included in the study. All strains had the 530 bp deletion in the ltx promoter region that is characteristic of the highly leukotoxic JP2 clone. Strains HK921 (=JP2), HK1199 and HK1200 were clinical isolates from young African-Americans suffering from aggressive periodontitis, and HK1519 and HK1651 were clinical isolates from immigrants in Sweden and Denmark originating from the Cape Verde Islands and Ghana, respectively. The complete genome sequence of strain HK1651 is available at http://www.genome.ou.edu/act.html. The remaining 20 clinical strains were isolated within one year from adolescents living in Morocco (Haubek et al., 2001). The strains were cultured on chocolate agar plates or in 2x YT medium (Sambrook et al., 1989) at 37 °C in air plus 5 % CO2.

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: 296–822 in pgi, 55–549 in recA, 32–590 in adk, 215–724 in frdB, 23–522 in atpG and 182–621 in mdh. For sequence analyses we used the program CLUSTALX (Thompson et al., 1997).


View this table:
[in this window]
[in a new window]
 
Table 1. Primers used for PCR amplification of internal fragments of six housekeeping genes, the six rrn operons and the seven IS150-like elements

Numbers indicate the region amplified in the HK1651 genome sequence.

 
MspI DNA fingerprinting.
Whole-cell DNA was prepared as previously described (Poulsen et al., 1988). Approximately 10 µg was digested with MspI, electrophoresed in a 1 % agarose gel overnight at 1·5 V cm–1 in Tris/acetate/EDTA (TAE) buffer, and visualized by staining with ethidium bromide (Sambrook et al., 1989).

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 24–534 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 ml–1, 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’.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains of the JP2 clone show minor differences
Five highly leukotoxic strains of A. actinomycetemcomitans, HK921 (JP2), HK1199, HK1200, HK1519 and HK1651, were analysed. The 530 bp deletion in the promoter of the ltx operon that is characteristic for the highly leukotoxic strains was verified in the five strains by PCR analysis, as previously described (Poulsen et al., 2003). Digestion of whole-cell DNA with MspI revealed identical DNA fingerprints (Fig. 1a). It should be noted that in the top part of the gel, the few separate bands each represent single DNA fragments in the size range >3 kb, whereas each of the visible bands in the lower part represents several DNA fragments migrating together. Thus, this technique only assays for the location of MspI restriction sites in a small fraction of the genome (approx. 5 %). Multilocus sequence typing (MLST) of internal fragments of six housekeeping genes, pgi, recA, adk, frdB, atpG and mdh, showed that the strains had an identical sequence in four of these gene fragments, that strain HK1651 had a single nucleotide substitution in the atpG gene fragment (C to G substitution at position 253 in the ORF), and that strain HK1199 had a substitution in the adk gene (A to G at position 361 in the ORF), as compared to the other strains. Thus, the five strains are genetically very similar and belong to the highly leukotoxic JP2 clone. However, Southern blot analyses of EcoRI-digested whole-cell DNA using 16S rRNA gene sequences or the IS150-like element as probes revealed additional differences among the strains (Fig. 1b, c). Both probes were amplified by PCR from genomic DNA of A. actinomycetemcomitans strain HK1651 and it should be noted that neither probe contains an EcoRI restriction site. In previous RFLP analyses of rrn genes in A. actinomycetemcomitans strains, we used the complete rrn operon from E. coli as probe, and therefore the hybridization patterns obtained here are not comparable to those previously obtained (Haubek et al., 1996, 1997). The EcoRI RFLP patterns indicated that all strains harboured six copies of the 16S rRNA gene and seven copies of the IS150-like element, which is in agreement with the complete genome sequence of strain HK1651. Strains HK921 and HK1519 showed identical patterns of hybridization with the two probes, whereas the other three strains differed in both RFLP analyses (Fig. 1b, c).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1. Genotyping of five strains of the JP2 clone of A. actinomycetemcomitans. Lane 1, HK1519; lane 2, HK921; lane 3, HK1651; lane 4, HK1200; lane 5, HK1199; lane 6, molecular weight marker (kb). (a) Whole-cell DNA was restricted with MspI, and DNA fragments were resolved by 1 % agarose gel electrophoresis and visualized by staining with ethidium bromide. (b) Southern blot analysis of EcoRI-restricted whole-cell DNA using 16S rRNA gene sequences as radioactively labelled probes. (c) As in (b), but using IS150-like sequences as probe. Note that the stronger bands in the Southern blot analyses represent two fragments migrating together.

 
The rrn genes and IS150-like elements are subject to intragenomic homologous recombination
We used the PCR method described by Helm & Maloy (2001) to demonstrate that the observed differences in EcoRI RFLP types among the highly leukotoxic strains are caused by intragenomic recombinations. Primers flanking each copy of the six rrn genes in the HK1651 genome were synthesized (Fig. 2a, Table 1). Each of the six ‘upstream’ primers was combined with each of the six ‘downstream’ ones and used in PCR of whole-cell DNA from each of the strains. The resulting PCR products were analysed by agarose gel electrophoresis. An example of the results (for strain HK1519) is shown in Fig. 2(b). Amplification of a DNA fragment of the expected size indicates that the two primers used flank an rrn gene copy in that genome. The extension product shares sequences with the other rrn genes, and therefore PCR jumping or bridging may occur; that is, short extension products may serve as primers on homologous regions of the other copies, and because a different combination of primer sites are now in cis, these products may subsequently be exponentially amplified by this set of primers. This is presumably the reason for the weak bands in the ‘negative’ primer combinations (Fig. 2b). In order to reduce this problem, the PCR included only 25 cycles. The results are summarized in Table 2. For strain HK1651, the primer combinations that amplified a strong band in the agarose gel analysis confirmed the arrangement of the six rrn genes in the genome sequence. For the other strains, a total of six combinations of primers amplified a strong band of the expected size. This is in agreement with the RFLP analysis, which indicated the presence of six 16S rRNA gene copies in each strain. However, all five strains differed in the primer combinations that resulted in a PCR product (Table 2). This can only be adequately explained by homologous recombination between non-allelic rrn gene copies. This results in genomic rearrangements.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. (a) Location of rrn genes and IS150-like sequence elements in the genome of strain HK1651. The locations were deduced from the genome sequence and the numbering is as in the databases, with the dnaA gene starting at position 1. The six rrn genes are indicated by black arrows and the seven IS150-like elements are indicated by open arrows. (b) Example of PCR analysis of the organization of rrn genes, showing results obtained for strain HK1519 with the 36 primer sets for the six rrn gene operons. (c) Example of PCR analysis of the organization of IS150-like elements, showing results for strain HK1651 with the 49 primer sets for the seven copies of IS150-like elements. Primer combinations that amplified a strong band of the expected size are indicated. Molecular weight (MW) markers (kb) are shown to the right.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Amplification of the rrn operons using flanking primers

Primer combinations giving a strong band in PCR are indicated by + and no/weak band by –.

 
The endonuclease I-CeuI specifically recognizes a 19 bp sequence in the 23S rRNA gene (Marshall & Lemieux, 1992). I-CeuI fingerprinting of the five genomes resolved by PFGE revealed six fragments for each strain, in accordance with the six rrn gene copies (Fig. 3). Homologous recombination between different rrn gene copies resulting in translocations or inversions in the genome does not influence the I-CeuI RFLP pattern. Four strains, HK1519, HK921, HK1200 and HK1199, showed identical fingerprints, whereas strain HK1651 differed in more bands. This suggests that in strain HK1651 homologous recombination within the genome has taken place between other multi-copy sequences, such as the IS150-like elements which are present on both sides of some of the rrn genes (see below).



View larger version (102K):
[in this window]
[in a new window]
 
Fig. 3. I-CeuI fingerprinting of strains of the JP2 clone of A. actinomycetemcomitans. Bacteria in agarose plugs were digested with I-CeuI and analysed by PFGE. Lane 1, HK1519; lane 2, HK921; lane 3, HK1651; lane 4, HK1200; lane 5, HK1199. Molecular weights (kb) are shown to the right.

 
The 49 combinations of primers flanking the seven IS150-like elements found in strain HK1651 were used in PCR of whole-cell DNA from the five strains analysed. An example of the results (for strain HK1651) is shown in Fig. 2(c). The results confirmed the arrangement of these elements in the genome sequence of strain HK1651 (Table 3). Also, for the remaining four strains, the number of successful amplifications was in agreement with the seven copies of the element detected by Southern blot analysis (Fig. 1c). Four strains, HK1519, HK921, HK1200 and HK1199, showed identical results in this PCR analysis. As described above, differences between HK1651 and the other strains in the combinations of primers giving rise to a strong band of the expected size in the PCR reflect homologous intragenomic recombination between different copies of the IS150-like element, which results in genomic rearrangements. The results indicate that a recombination between IS150-2 and IS150-4 that is present in different orientations and surrounding rrn3 has taken place in strain HK1651, as compared to the other four strains. This may explain the differences observed in the I-CeuI fingerprints.


View this table:
[in this window]
[in a new window]
 
Table 3. Amplification of the IS150-like elements using flanking primers

Primer combinations giving a strong band in PCR are indicated by + and no/weak band by –.

 
XhoI fingerprints resolved by PFGE differ among strains of the JP2 clone
Genomic DNA fingerprinting using a rare-cutting restriction enzyme combined with PFGE to resolve the resulting fragments would be influenced by genomic rearrangements, provided that the rearranged fragment harbours a site for the enzyme used. Accordingly, the XhoI DNA fingerprints of the five strains analysed showed four distinct patterns (Fig. 4a). The five strains were isolated from persons originating from different parts of Africa (see Methods). In addition, 20 strains assigned to the highly leukotoxic JP2 clone and isolated from adolescents living in Morocco were typed. For these 20 strains, the presence of the 530 bp deletion in the ltx promoter was verified by PCR. In addition, whole-cell DNA fingerprinting using the restriction enzyme MspI revealed that all of the strains showed a pattern identical to that of the five strains described above and shown in Fig. 1(a). Among the 20 strains, a total of eight different XhoI profiles were detected, with one prevailing type represented by nine strains. Examples of XhoI DNA fingerprints are shown in Fig. 4(b).



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 4. XhoI DNA fingerprinting of strains of the JP2 clone of A. actinomycetemcomitans. Bacteria in agarose plugs were digested with XhoI and the resulting DNA fragments resolved by PFGE. (a) Lane 1, HK1519; lane 2, HK921; lane 3, HK1651; lane 4, HK1200; lane 5, HK1199. (b) Examples of XhoI fingerprints obtained for strains isolated from Moroccan adolescents. Lanes 1 and 2 represent the prevailing type. Molecular weight markers (kb) are shown to the right.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The presence of multi-copy sequence elements interspersed in the genome opens up the possibility of homologous recombination within the genome (Krawiec & Riley, 1990; Liu & Sanderson, 1996; Hughes, 2000). For two elements present in the same orientation, such an intragenomic recombination may result in the deletion of the intervening region, including loss of one copy of the element, and if the circular deleted segment inserts into the chromosome by homologous recombination with a different copy of the element, the result is a transposition. Recombinations between non-allelic repeat elements in the same orientation present on sister chromosomes after replication can lead to duplication of the region bounded by these elements. For elements in opposite orientations, the result of a homologous recombination will be an inversion of the intervening region. Here, we show by a PCR method that the rRNA genes and IS150-like elements are subject to intragenomic recombination in members of the highly leukotoxic JP2 clone of A. actinomycetemcomitans. This results in rearrangements within the genome. Genomic deletions or duplications seem not to have taken place in the five analysed members of the JP2 clone, since the MspI DNA fingerprints look alike, and because the number of rrn genes and IS150-like elements is conserved among the strains. For both the six rrn genes and the seven IS150-like elements, copies are found in both orientations in the genome of strain HK1651 (Fig. 2a). Thus, the genomic rearrangements in the JP2 clone resulting from homologous recombination between members of these two multi-copy sequences may include inversions and transpositions. Notably, such rearrangements do not change the gene content of the bacterium.

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.


   ACKNOWLEDGEMENTS
 
We thank M. Kilian for helpful discussions and for comments on the manuscript and U. S. Sørensen for kind help with the figures. We are grateful to B. A. Roe, F. Z. Najar, A. Gillaspy, S. Clifton, T. Ducey, L. Lewis and D. W. Dyer for the use of the unpublished genome sequence from the Actinobacillus Genome Sequencing Project at the University of Oklahoma. This study was supported by grants 22-01-0265 ct/mp and 22-02-0306 ch/mp from the Danish Medical Research Council.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brogan, J. M., Lally, E. T., Poulsen, K., Kilian, M. & Demuth, D. R. (1994). Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains. Infect Immun 62, 501–508.[Abstract]

Bueno, L. C., Mayer, M. P. & DiRienzo, J. M. (1998). Relationship between conversion of localized juvenile periodontitis-susceptible children from health to disease and Actinobacillus actinomycetemcomitans leukotoxin promoter structure. J Periodontol 69, 998–1007.[Medline]

Contreras, A., Rusitanonta, T., Chen, C., Wagner, W. G., Michalowicz, B. S. & Slots, J. (2000). Frequency of 530-bp deletion in Actinobacillus actinomycetemcomitans leukotoxin promoter region. Oral Microbiol Immunol 15, 338–340.[CrossRef][Medline]

Hansen, S. M., Uldbjerg, N., Kilian, M. & Sørensen, U. B. S. (2004). Dynamics of Streptococcus agalactiae colonization in women during and after pregnancy and in their infants. J Clin Microbiol 42, 83–89.[Abstract/Free Full Text]

Haraszthy, V. I., Hariharan, G., Tinoco, E. M. B., Cortelli, J. R., Lally, E. T., Davis, E. & Zambon, J. J. (2000). Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. J Periodontol 71, 912–922.[CrossRef][Medline]

Haubek, D., Poulsen, K., Westergaard, J., Dahlen, G. & Kilian, M. (1996). Highly toxic clone of Actinobacillus actinomycetemcomitans in geographically widespread cases of juvenile periodontitis in adolescents of African origin. J Clin Microbiol 34, 1576–1578.[Abstract]

Haubek, D., DiRienzo, J. M., Tinoco, E. M., Westergaard, J., Lopez, N. J., Chung, C. P., Poulsen, K. & Kilian, M. (1997). Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis. J Clin Microbiol 35, 3037–3042.[Abstract]

Haubek, D., Ennibi, O. K., Poulsen, K., Poulsen, S., Benzarti, N. & Kilian, M. (2001). Early-onset periodontitis in Morocco is associated with the highly leukotoxic clone of Actinobacillus actinomycetemcomitans. J Dent Res 80, 1580–1583.[Abstract/Free Full Text]

Haubek, D., Ennibi, O. K., Poulsen, K., Benzarti, N. & Baelum, V. (2004). The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans and progression of periodontal attachment loss. J Dent Res 83, 767–770.[Abstract/Free Full Text]

Hayashida, H., Poulsen, K., Takagi, O. & Kilian, M. (2000). Phylogenetic associations of ISAa1 and IS150-like insertion sequences in Actinobacillus actinomycetemcomitans. Microbiology 146, 1977–1985.[Medline]

Hayashida, H., Poulsen, K. & Kilian, M. (2002). Differences in iron acquisition from human haemoglobin among strains of Actinobacillus actinomycetemcomitans. Microbiology 148, 3993–4001.[Medline]

Helm, R. A. & Maloy, S. (2001). Rapid approach to determine rrn arrangement in Salmonella serovars. Appl Environ Microbiol 67, 3295–3298.[Abstract/Free Full Text]

Hughes, D. (2000). Evaluating genome dynamics: the constraints on rearrangements within bacterial genomes. Genome Biol 1, 0006.1–000006.8.

Krawiec, S. & Riley, M. (1990). Organization of the bacterial chromosome. Microbiol Rev 54, 502–539.[Medline]

Liu, S.-L. & Sanderson, K. E. (1996). Highly plastic chromosomal organization in Salmonella typhi. Proc Natl Acad Sci U S A 93, 10303–10308.[Abstract/Free Full Text]

Marshall, P. & Lemieux, C. (1992). The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene. Nucleic Acids Res 20, 6401–6407.[Abstract]

Poulsen, K., Hjorth, J. P. & Kilian, M. (1988). Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains. Infect Immun 56, 987–992.[Medline]

Poulsen, K., Ennibi, O. K. & Haubek, D. (2003). Improved PCR for detection of the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans in subgingival plaque samples. J Clin Microbiol 41, 4829–4832.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.[CrossRef]

Tsai, C.-C., Shenker, B. J., DiRienzo, J. M., Malamud, D. & Taichman, N. S. (1984). Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B. Infect Immun 43, 700–770.[Medline]

Received 12 May 2005; revised 1 July 2005; accepted 11 July 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Eriksen, K. T.
Articles by Poulsen, K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Eriksen, K. T.
Articles by Poulsen, K.
Agricola
Articles by Eriksen, K. T.
Articles by Poulsen, K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.