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Journal of Bacteriology, January 2005, p. 567-575, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.567-575.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans

Kai Soo Tan,1 Grace Ong,2 and Keang Peng Song1*

Microbial Pathogenesis Laboratory, Department of Microbiology, Faculty of Medicine,1 Department of Preventive Dentistry, Faculty of Dentistry, National University of Singapore, Singapore2

Received 4 August 2004/ Accepted 13 October 2004

In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria. Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within the cdt gene of A. actinomycetemcomitans. By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of the cdt RNA was located within the cdtC gene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations.

* Corresponding author. Mailing address: Department of Microbiology, National University of Singapore, 5 Science Dr. 2, Singapore 117597. Phone: 65-6874 3278. Fax: 65-6776 6872. E-mail: micskp{at}nus.edu.sg.

Journal of Bacteriology, January 2005, p. 567-575, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.567-575.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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