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J Med Microbiol 56 (2007), 1536-1548; DOI: 10.1099/jmm.0.47289-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants

Keiji Nagano1,{dagger}, Yukitaka Murakami1, Kiyoshi Nishikawa1, Junpei Sakakibara1,2, Kazuo Shimozato2 and Fuminobu Yoshimura1

1 Department of Microbiology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan

2 Oral and Maxillofacial Surgery II, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan

Correspondence
Yukitaka Murakami
yukimu{at}dpc.agu.ac.jp

Received 14 March 2007
Accepted 24 July 2007

The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the {Delta}ragA mutant reduced RagB expression considerably and the {Delta}ragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the {Delta}ragA and {Delta}ragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the {Delta}ragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein–protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.

Abbreviations: CAT, chloramphenicol acetyltransferase; CBB, Coomassie brilliant blue; DAB, 3,3'-diaminobenzidine.

{dagger}Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.

The GenBank/EMBL/DDBJ accession number for the P. gingivalis ATCC 33277T ragA, ragB and flanking region sequence is AB205195.






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