Verification of a topology model of PorT as an integral outer-membrane protein in Porphyromonas gingivalis

• Published in: Microbiology (Society for General Microbiology) Vol. 155; no. 2; pp. 328 - 337
• Main Authors: Nguyen, Ky-Anh, Zylicz, Jasiek, Szczesny, Pawel, Sroka, Aneta, Hunter, Neil, Potempa, Jan
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.02.2009; Society for General Microbiology
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacteriology; Biological and medical sciences; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Fundamental and applied biological sciences. Psychology; Microbiology; Morphology, structure, chemical composition; Mutagenesis, Insertional; Mutation; Porphyromonas gingivalis - chemistry; Porphyromonas gingivalis - genetics; Porphyromonas gingivalis - metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport; Porphyromonas gingivalis; Membrane protein; Molecular structure; Bacteria; Models; External membrane; Bacteroidaceae
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.024323-0

1 Institute of Dental Research, Westmead Centre for Oral Health and Westmead Millennium Institute, Sydney, Australia 2 Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 3 Department of Bioinformatics, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland 4 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA Correspondence Ky-Anh Nguyen kyanhn{at}gmail.com PorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases, the gingipains, from the periodontal pathogen Porphyromonas gingivalis . It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel, membrane-traversing β -strands. In this report, the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6 x His) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly, those PorT mutations adversely affecting gingipain secretion enhanced expression of the porT gene but at the same time suppressed the transcription of the gingipain rgpB gene. Further, PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of the Bacteroidetes phylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins. Abbreviations: AP, alkaline phosphatase; CTD, C-terminal domain; IM, inner membrane; OM, outer membrane; TLCK, tosyl- L -lysine chloromethyl ketone A supplementary table of primers and three supplementary figures are available with the online version of this paper.