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Infection and Immunity, July 2000, p. 4012-4017, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of ragAB as a Temperature-Regulated Operon of Porphyromonas gingivalis W50 Using Differential Display of Randomly Primed RNA

William A. Bonass,1,* Philip D. Marsh,1 Rimondia S. Percival,1 Joseph Aduse-Opoku,2 Shirley A. Hanley,2 Deirdre A. Devine,1 and Michael A. Curtis2

Oral Microbiology Group, Division of Oral Biology, Leeds Dental Institute, University of Leeds, LS2 9LU,1 and MRC Molecular Pathogenesis Group, Department of Medical Microbiology, St. Bartholomew's and Royal London School of Medicine and Dentistry, London E1 2AA,2 United Kingdom

Received 7 December 1999/Returned for modification 16 February 2000/Accepted 31 March 2000

Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41°C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

* Corresponding author. Mailing address: Oral Microbiology Research Group, Division of Oral Biology, Leeds Dental Institute, University of Leeds, Leeds LS2 9LU, United Kingdom. Phone: 44 113 2336184. Fax: 44 113 2336165. E-mail: W.A.Bonass{at}leeds.ac.uk.

Infection and Immunity, July 2000, p. 4012-4017, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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