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Identification of a New Membrane-associated Protein That Influences Transport/Maturation of Gingipains and Adhesins of Porphyromonas gingivalis

2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m413544200

1083-351X

Keiko Sato, Eiko Sakai, Paul D. Veith, Mikio Shoji, Yuichiro Kikuchi, Hideharu Yukitake, Naoya Ohara, Mariko Naito, Kuniaki Okamoto, Eric C. Reynolds, Koji Nakayama,

Bacterial biofilms and quorum sensing

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp′-′rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured. The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp′-′rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured. Periodontal disease, the major cause of tooth loss in the general population of industrial nations (1Papapanou P.N. J. Int. Acad. Periodontol. 1999; 1: 110-116PubMed Google Scholar, 2Irfan U.M. Dawson D.V. Bissada N.F. J. Int. Acad. Periodontol. 2001; 3: 14-21PubMed Google Scholar), is a chronic inflammatory disease of the periodontium that leads to erosion of the attachment apparatus and supporting bone for the teeth (3Armitage G.C. Ann. Periodontol. 1996; 1: 37-215Crossref PubMed Scopus (233) Google Scholar) and is one of the most common infectious diseases of humans (4Oliver R.C. Brown L.J. Loe H. J. Periodontol. 1998; 69: 269-278Crossref PubMed Scopus (327) Google Scholar). The obligately anaerobic Gram-negative bacterium Porphyromonas gingivalis has become recognized as a major pathogen for adult periodontitis (5Christersson L.A. Fransson C.L. Dunford R.G. Zambon J. J. Periodontol. 1992; 63: 418-425Crossref PubMed Scopus (103) Google Scholar). The microorganism possesses several potential virulence factors for periodontopathogenicity (6Holt S.C. Kesavalu L. Walker S. Genco C.A. Periodontol. 2000. 1999; 20: 168-238Crossref PubMed Scopus (516) Google Scholar). Among these factors the proteolytic enzymes are of special importance, since some of them have the abilities to destroy periodontal tissue directly or indirectly (7Lawson D.A. Meyer T.F. Infect. Immun. 1992; 60: 1524-1529Crossref PubMed Google Scholar, 8Smalley J.W. Birss A.J. Shuttleworth C.A. Arch. Oral Biol. 1988; 33: 323-329Crossref PubMed Scopus (62) Google Scholar). P. gingivalis produces large amounts of lysine-specific (Lys-gingipain, Kgp) and arginine-specific (Arg-gingipain, Rgp) cysteine proteinase on the cell surface and in the extracellular milieu (9Pike R. McGraw W. Potempa J. Travis J. J. Biol. Chem. 1994; 269: 406-411Abstract Full Text PDF PubMed Google Scholar, 10Rangarajan M. Smith S.J.M.U.S. Curtis M.A. Biochem. J. 1997; 323: 701-709Crossref PubMed Scopus (64) Google Scholar, 11Bhogal P.S. Slakeski N. Reynolds E.C. Microbiology. 1997; 143: 2485-2495Crossref PubMed Scopus (69) Google Scholar, 12Slakeski N. Bhogal P.S. O'Brien-Simpson N.M. Reynolds E.C. Microbiology. 1998; 144: 1583-1592Crossref PubMed Scopus (59) Google Scholar). The Kgp and Rgp proteinases have the ability to specifically cleave substrates on the carboxyl side of lysine and arginine, respectively. Kgp and Rgp are encoded by one gene (kgp) and two separate genes (rgpA and rgpB) on the P. gingivalis chromosome, respectively and are widely implicated as important virulence factors in the pathogenesis of periodontal disease (13Potempa J. Banbula A. Travis J. Periodontol. 2000. 2000; 24: 153-192Crossref PubMed Scopus (276) Google Scholar, 14Curtis M.A. Kuramitsu H.K. Lanz M. Macrine F.L. Nakayama K. Potempa J. Reynolds E.C. Aduse-Opoku J. J. Periodont. Res. 1999; 34: 464-472Crossref PubMed Scopus (177) Google Scholar). The kgp and rgpA genes having 5,193- and 5,118-bp open reading frames (ORFs) 1The abbreviations used are: ORFs, open reading frames; PBS, phosphate-buffered saline; CBB, Coomassie Brilliant Blue; BSA, bovine serum albumin; BHI, brain heart infusion; Cm, chloramphenicol; Em, erythromycin; Tc, tetracycline; OMF, outer membrane factor; α-KG, α-ketoglutarate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. encode proteins that consist of four domains: signal sequence, propeptide, mature proteinase, and C-terminal adhesin domains. The C-terminal adhesin domain region that is thought to be involved in hemagglutination comprises four subdomains. The rgpB gene has a 2,208-bp ORF, the amino acid sequence of which is similar to that of rgpA, but lacks most of the adhesin domain. These proteinases are synthesized as pre-proenzymes that are processed and secreted into the extracellular milieu as the mature proteinases or located on the cell surface as complexes non-covalently associated with the adhesin domain proteins; however, the precise mechanism of the transport/maturation is still unknown. Previous studies have shown a link between colonial pigmentation on blood agar plates, hemagglutination and Kgp/Rgp activity in P. gingivalis cells (15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 16Chen T. Dong H. Yong R. Duncan M.J. Microb. Pathog. 2000; 28: 235-247Crossref PubMed Scopus (35) Google Scholar). P. gingivalis wild-type strains form black-pigmented colonies resulting from accumulation of the oxidized form of heme on the cell surface (17Shah H.N. Gharbia S.E. FEMS Microbiol. Lett. 1989; 52: 213-217Crossref PubMed Scopus (44) Google Scholar, 18Smalley J.W. Silver J. Marsh P.J. Birss A.J. Biochem. J. 1998; 331: 681-685Crossref PubMed Scopus (104) Google Scholar), but the Kgp-null mutants exhibit reduced pigmentation and the Kgp/Rgp-null mutants show no pigmentation (15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 19Okamoto K. Nakayama K. Kadowaki T. Abe N. Ratnayake D.B. Yamamoto K. J. Biol. Chem. 1998; 273: 21225-21231Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It suggests that these proteinases play an important role in acquisition of heme from erythrocytes (20Nakayama K. Ratnayake D.B. Tsukuba T. Kadowaki T. Yamamoto K. Fujimura S. Mol. Microbiol. 1998; 27: 51-61Crossref PubMed Scopus (102) Google Scholar, 21Lewis J.P. Dawson J.A. Hannis J.C. Muddiman D. Macrina F.L. J. Bacteriol. 1999; 181: 4905-4913Crossref PubMed Google Scholar). Transposon mutagenesis has been applied to the isolation of pigment-less mutants of P. gingivalis by several researchers (16Chen T. Dong H. Yong R. Duncan M.J. Microb. Pathog. 2000; 28: 235-247Crossref PubMed Scopus (35) Google Scholar, 22Genco C.A. Schifferle R.E. Njoroge T. Forng R.Y. Cutler C.W. Infect. Immun. 1995; 63: 393-401Crossref PubMed Google Scholar, 23Genco C.A. Simpson W. Forng R.Y. Egal M. Odusanya B.M. Infect. Immun. 1995; 63: 2459-2466Crossref PubMed Google Scholar, 24Simpson W. Wang C.Y. Mikolajczyk-Pawlinska J. Potempa J. Travis J. Bond V.C. Genco C.A. Infect. Immun. 1999; 67: 5012-5020Crossref PubMed Google Scholar). Chen et al. (16Chen T. Dong H. Yong R. Duncan M.J. Microb. Pathog. 2000; 28: 235-247Crossref PubMed Scopus (35) Google Scholar) isolated non-pigmented mutants that had the transposon Tn4351 DNA within kgp. In addition, Simpson et al. (24Simpson W. Wang C.Y. Mikolajczyk-Pawlinska J. Potempa J. Travis J. Bond V.C. Genco C.A. Infect. Immun. 1999; 67: 5012-5020Crossref PubMed Google Scholar) found that a non-pigmented mutant has the insertion sequence element IS1126 at the promoter locus of kgp. These results confirmed the involvement of kgp in pigmentation. Recently, non-kgp mutations causing no pigmentation have been found (16Chen T. Dong H. Yong R. Duncan M.J. Microb. Pathog. 2000; 28: 235-247Crossref PubMed Scopus (35) Google Scholar, 25Abaibou H. Chen Z. Olango G.J. Liu Y. Edwards J. Fletcher H.M. Infect. Immun. 2001; 69: 325-335Crossref PubMed Scopus (54) Google Scholar). Chen et al. (16Chen T. Dong H. Yong R. Duncan M.J. Microb. Pathog. 2000; 28: 235-247Crossref PubMed Scopus (35) Google Scholar) found that Tn4351 was inserted into a putative glycosyl (rhamnosyl) transferase-encoding gene in several non-pigmented mutants and Abaibou et al. (25Abaibou H. Chen Z. Olango G.J. Liu Y. Edwards J. Fletcher H.M. Infect. Immun. 2001; 69: 325-335Crossref PubMed Scopus (54) Google Scholar) found that the gene vimA located downstream of recA has a role in pigmentation. Recently, we have found that the gene porR, which is located at a gene cluster for glycan biosynthesis is involved in the biosynthesis of cell surface polysaccharide that may function as the anchor for Rgp and Kgp, via attachment to the C-terminal adhesins (26Shoji M. Ratnayake D.B. Shi Y. Kadowaki T. Yamamoto K. Yoshimura F. Akamine A. Curtis M.A. Nakayama K. Microbiology. 2002; 148: 1183-1191Crossref PubMed Scopus (80) Google Scholar). In this study, we isolated a non-pigmented mutant that has an insertion mutation within the new gene porT and found that the unprocessed gene products of kgp, rgpA, and rgpB are accumulated in the periplasmic space of the porT mutant cells. Strains and Culture Conditions—All P. gingivalis strains used are shown in Table I. P. gingivalis cells were grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched brain heart infusion (BHI) medium, and on enriched tryptic soy agar (28Nakayama K. Kadowaki T. Okamoto K. Yamamoto K. J. Biol. Chem. 1995; 270: 23619-23626Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). For blood agar plates, defibrinated laked sheep blood was added to enriched tryptic soy agar at 5%. As a defined minimal medium, we used α-ketoglutarate/bovine serum albumin (α-KG/BSA) medium for the growth of P. gingivalis (29Milner P. Batten J.E. Curtis M.A. FEMS Microbiol. Lett. 1996; 140: 125-130PubMed Google Scholar). For selection and maintenance of the antibiotic-resistant strains, antibiotics were added to the medium at the following concentrations: ampicillin, 50 μg/ml; kanamycin, 50 μg/ml; chloramphenicol (Cm), 20 μg/ml; erythromycin (Em), 10 μg/ml; and tetracycline (Tc), 0.7 μg/ml.Table IP. gingivalis strains used in this studyNameGenotypeSource or ref.33277Wild typeATCCKDP106porT::Tn4351This studyKDP107porR::kan ermF26Shoji M. Ratnayake D.B. Shi Y. Kadowaki T. Yamamoto K. Yoshimura F. Akamine A. Curtis M.A. Nakayama K. Microbiology. 2002; 148: 1183-1191Crossref PubMed Scopus (80) Google ScholarKDP108porR+ ermF26Shoji M. Ratnayake D.B. Shi Y. Kadowaki T. Yamamoto K. Yoshimura F. Akamine A. Curtis M.A. Nakayama K. Microbiology. 2002; 148: 1183-1191Crossref PubMed Scopus (80) Google ScholarKDP112rgpA1::tetQ rgpB1::ermF15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google ScholarKDP117porT1::kan ermFThis studyKDP118porT+ ermFThis studyKDP129kgp-2::cat15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google ScholarKDP136kgp-2::cat rgpA2::[ermF ermAM] rgpB2::tetQ15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google ScholarKDP150fimA::[ermF ermAM]27Shoji M. Naito M. Yukitake H. Sato K. Sakai E. Ohara N. Nakayama K. Mol. Microbiol. 2004; 52: 1513-1525Crossref PubMed Scopus (65) Google ScholarKDP350porT1::kan ermF fimA::[porT+ tetQ]This studyKDP351kgp-2::cat porT2::[ermF ermAM]This studyKDP352kgp-2::cat fimA::[kgp′-′rgpB tetQ]This studyKDP353kgp-2::cat porT2::[ermF ermAM] fimA::[kgp′-′rgpB tetQ]This study Open table in a new tab Transposon Mutagenesis and Gene-directed Mutagenesis—Transposon mutagenesis of P. gingivalis strain 33277 (ATCC) with Tn4351 and gene-directed mutagenesis of P. gingivalis strains with electroporation were described previously (26Shoji M. Ratnayake D.B. Shi Y. Kadowaki T. Yamamoto K. Yoshimura F. Akamine A. Curtis M.A. Nakayama K. Microbiology. 2002; 148: 1183-1191Crossref PubMed Scopus (80) Google Scholar). Construction of Plasmids and Bacterial Strains—A PvuII DNA fragment (8 kb) containing Tn4351 DNA in the chromosomal DNA of P. gingivalis KDP106 was cloned into the HincII region of pACYC184 (30Rose R.E. Nucleic Acids Res. 1988; 16: 355Crossref PubMed Scopus (329) Google Scholar). The resulting plasmid was digested with AvaI and the larger AvaI fragment self-ligated to yield pKD263. The kanamycin-resistance gene (kan) block (1.3 kb) of pUC4K (31Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3784) Google Scholar) was inserted into a unique SalI site within the porT gene of pKD263, resulting in pKD304. The NcoI-EcoRI fragment of pKD263 that contained the porT gene disrupted with the kan DNA block was then inserted into the PvuII site of pKD283, an EcoRI-fragment-deleted derivative of pMJF-2 (32Feldhaus M.J. Hwa V. Cheng Q. Salyers A.A. J. Bacteriol. 1991; 173: 4540-4543Crossref PubMed Google Scholar), resulting in pKD305. P. gingivalis 33277 was transformed to be Em-resistant (Emr) by electroporation with pKD305, resulting in KDP117 (porT1::kan ermF) and KDP118 (porT+ermF). A porT region DNA (1 kb) was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides 5′-TATTGTTGTGAGGTAGGTTATGC-3′ and 5′-GCTCTAGAAATATCCAAAAAGCTTAGGCGTCG-3′, digested with EcoRI and inserted into a unique EcoRI site of pKD713, a derivative of pKD703 (32Feldhaus M.J. Hwa V. Cheng Q. Salyers A.A. J. Bacteriol. 1991; 173: 4540-4543Crossref PubMed Google Scholar) containing the tetQ DNA block of pKD375 (15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) at the BamHI site, resulting in pKD850 (fimA::[porT+tetQ]). KDP117 was then transformed with the NotI-linearized DNA of pKD850 to yield KDP350 (porT1::kan ermF fimA::[porT+tetQ]). For construction of a plasmid containing the erm DNA block between a porT-upstream DNA and a porT-downstream DNA, the porT-upstream DNA region encoding PG0750 was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5′-TAGGATCCTAGTTGTCACGCTCTTTTCGAC-3′ and 5′-TAGGTCGACAGCGCTTGCGGCGGAAAAAGAAG-3′, cloned into the pGEM-T Easy vector (Promega) and digested with SpeI and BamHI. The resulting DNA fragment was then inserted into the corresponding region of pKD355, a derivative of pBluescript II SK(-) carrying the erm DNA block (15Shi Y. Ratnayake D.B. Okamoto K. Abe N. Yamamoto K. Nakayama K. J. Biol. Chem. 1999; 274: 17955-17960Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) between the BamHI and EcoRI sites, resulting in pKD356. The porT-downstream DNA region encoding PG0752 was PCR-amplified using a pair of oligonucleotides, 5′-GTGAATTCCCTGAGAGAATAATCTTCAATTCT-3′ and 5′-GCGGCCGCTATACAGGATCGTATTGAGTGCT-3′, cloned into the pGEM-T Easy vector and digested with EcoRI. The resulting DNA fragment was inserted into the corresponding region of pKD356 to yield pKD357 (porT2::[ermF ermAM]). P. gingivalis KDP129 (kgp::cat) was then transformed with the NotI-linearized pKD357 DNA to yield KDP351 (kgp::cat porT2::[ermF ermAM]). For construction of a kgp′-′rgpB chimera gene, the DNA (0.6 kb) of the rgpB gene encoding the C-terminal domain was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5′-GCTCTAGAAGAAACGAACTTGACGCTCACCGTA-3′ and 5′-CATAAACGACTGCAATGCAACGGCGGCCGCA-5′. The amplified DNA was digested with EagI and XbaI, and inserted into the corresponding region of pKD851, a derivative of pBluescript II SK(-) that contained the His (X6)-tag DNA between BamHI and XbaI, resulting in pKD852. The middle region of the kgp gene was PCR-amplified from the chromosomal DNA of 33277 using a pair of oligonucleotides, 5′-TACTCGAGCTTATCGTGCAATGCCTAAGACC-3′ and 5′-CGGATCCAATACATCGTTTGCAGGTTCGATCG-3′, digested with XhoI and BamHI. The resulting XhoI-BamHI fragment was then inserted into the corresponding region of pKD852 to yield pKD853. The 2.6-kb AvaI DNA fragment encoding the signal peptide, propeptide, and mature proteinase portions of the kgp gene was isolated from the pNKV (33Okamoto K. Kadowaki T. Nakayama K. Yamamoto K. J. Biochem. (Tokyo). 1996; 120: 398-406Crossref PubMed Scopus (96) Google Scholar) and inserted into the AvaI site of pKD853 to yield pKD854 (kgp′-′rgpB). The KpnI-NotI DNA fragment of pKD854 containing the kgp′-′rgpB chimera gene DNA was inserted into the corresponding region of pKD713, resulting in pKD855 (fimA::[kgp′-′rgpB tetQ]). For construction of P. gingivalis strains possessing the kgp′-′rgpB chimera gene, KDP129 (kgp::cat) and KDP351 (kgp::cat porT2::[ermF ermAM]) were transformed with the NotI-linearized pKD855 DNA to yield KDP352 (kgp::cat fimA::[kgp′-′rgpB tetQ]) and KDP353 (kgp::cat porT2::[ermF ermAM] fimA::[kgp′-′rgpB tetQ]), respectively. Hemagglutination Assay—Overnight cultures of P. gingivalis strains in enriched BHI medium were centrifuged, washed with PBS, and resuspended in PBS. The bacterial suspensions were then diluted in a 2-fold series with PBS. A 100-μl aliquot of each suspension was mixed with an equal volume of human erythrocyte suspension (1% in PBS) and incubated in a round bottom microtiter plate at room temperature for 3 h. Enzymatic Assays—Kgp and Rgp activities were determined using the synthetic substrates t-butyl-oxycarbonyl-l-valyl-l-leucyl-l-lysine-4-methyl-7-coumarylamide (Boc-Val-Leu-Lys-MCA) (final concentration 20 μm) and carbobenzoxy-l-phenyl-l-arginine-4-methyl-7-coumarylamide (Z-Phe-Arg-MCA) in 20 mm sodium phosphate buffer (pH 7.5) containing 5 mm cysteine in a total volume of 1 ml. After incubation at 40 °C for 10 min, the reaction was terminated by adding 1 ml of 10 mm iodoacetamide (pH 5.0), and the released 7-amino-4-methylcoumarin was measured at 460 nm (excitation at 380 nm). One unit of enzyme activity was defined as the amount of enzyme required to release 1 nmol of 7-amino-4-methylcoumarin/ml under these conditions. Northern Blot Analysis—Total RNA was extracted from P. gingivalis cells grown to mid-exponential phase (OD600, 0.3) using an RNA purification kit (RNeasy Protect minikit, Qiagen). 5 μg of RNA were electrophoresed in 1.2% agarose gel and then transferred to a nylon membrane (Hybond-N, Amersham Biosciences) according to the method described by Sambrook et al. (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor1989: 7.39-7.49Google Scholar). The antisense mRNA probes specific for the 0.5-kb BstXI(T325)-SphI(M482) region of rgpB and the 0.5-kb AccI(T346)-EcoRI(E510) region of kgp were constructed by using the pSPUTK plasmid (Stratagene). The RNA probes were labeled with digoxigenin using the DIG RNA labeling kit (Roche Applied Science). Northern blot hybridization and detection were carried out according to the manufacturer's recommendation. Subcellular Fractionation—Subcellular fractionation of P. gingivalis cells was performed essentially according to Murakami et al. (35Murakami Y. Imai M. Nakamura H. Yoshimura F. Eur. J. Oral Sci. 2002; 110: 157-162Crossref PubMed Scopus (56) Google Scholar). Briefly, P. gingivalis cells from a 3,000-ml culture were harvested by centrifugation at 10,000 × g for 30 min at 4 °C, and resuspended with 100 ml of PBS containing 0.1 mmNα-p-tosyl-l-lysine chloromethyl ketone (TLCK), 0.1 mm leupeptin, and 0.5 mm EDTA. The cells were disrupted in a French pressure cell at 100 MPa by two passes. The remaining intact bacterial cells were removed by centrifugation at 2,400 × g for 10 min, and the supernatant was subjected to ultracentrifugation at 100,000 × g for 60 min. The pellets were treated with 1% Triton X-100 in PBS containing 20 mm MgCl2 for 30 min at 20 °C. The outer membrane fraction was recovered as a precipitate by ultracentrifugation at 100,000 × g for 60 min at 4 °C. The supernatant was obtained as the inner membrane fraction. DEAE-Sepharose Chromatography and Affinity Chromatography— Cells of P. gingivalis KDP117 (porT) from a 500-ml culture were harvested by centrifugation at 10,000 × g for 30 min at 4 °C and resuspended with 20 mm phosphate buffer (pH 7.0) containing 0.1 mm TLCK, 0.1 mm leupeptin and 0.5 mm EDTA, and sonicated. Unbroken cells and large debris were removed by centrifugation (1,000 × g, 30 min, 4 °C), and the cloudy supernatant was applied to a column (2.6 × 40 cm) of DEAE Sepharose (Sepharose CL-6B, Amersham Biosciences), which had been equilibrated with 20 mm phosphate buffer (pH 7.0). After being washed thoroughly with the same buffer, proteins were eluted stepwise with 100 ml of the same buffer containing 100, 200, 300, 400, 500, and 700 mm NaCl at a flow rate of 0.5 ml/min. Proteins with high molecular masses (>150 kDa) were detected only in the 200 mm NaCl eluent. The 200 mm NaCl eluent, which immunoreacted with anti-Hgp44, was dialyzed against 0.1 m NaHCO3 (pH 8.3) and applied to a column of BrCN-activated Sepharose 4B (Amersham Biosciences) conjugated with anti-Hgp44 IgG, which had been equilibrated with the same buffer. The column was then eluted with 0.1 m Gly-HCl (pH 2.8), 0.5 m NaCl. The eluent was immediately equilibrated with 1 m Tris-HCl (pH 9.0). The kgp′-′rgpB chimera gene product was purified using a resin precharged with Ni2+ (ProBond™ resin, Invitrogen). Briefly, P. gingivalis KDP353 cells (50 ml culture) were resuspended in 8 ml of the guanidinium lysis buffer (6 m guanidine HCl, 20 mm sodium phosphate (pH 7.8), 500 mm NaCl) and slowly rocked for 10 min at room temperature. After centrifugation at 3,000 × g for 15 min, the supernatant was applied to the resin column, which had been equilibrated with the denaturing binding buffer (8 m urea, 20 mm sodium phosphate, pH 7.8, 500 mm NaCl). The column was washed with the denaturing wash buffer (8 m urea, 20 mm sodium phosphate, pH 6.0, 500 mm NaCl) and then eluted with the denaturing elution buffer (8 m urea, 20 mm sodium phosphate, pH 4.0, 500 mm NaCl). The resulting fractions were analyzed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB). These proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and stained with CBB. The protein band migrating to the position corresponding to a molecular mass of 78 kDa was cut out and subjected to N-terminal amino acid sequencing with an automatic protein sequencer (protein sequencing system LF3600D, Beckman). Preparation of Anti-Hgp44, Anti-HbR, and Anti-PorT Antisera— Recombinant Hgp44 protein was obtained as described previously (36Kamaguchi A. Ohyama T. Sakai E. Nakamura R. Watanabe T. Baba H. Nakayama K. Microbiology. 2003; 149: 1257-1264Crossref PubMed Scopus (50) Google Scholar). A peptide derived from the amino acid sequence (Thr221 to Leu233) of PorT with an N-terminal cysteine residue, CTHERPDLLDDYKL, which was conjugated to keyhole limpet hemocyanin was purchased from Sigma Genosys. The recombinant Hgp44 protein and the conjugated PorT peptide were mixed with Freund's complete adjuvant and injected subcutaneously into rabbits (Japan White) with two booster shots of a mixture of these antigens and Freund's incomplete adjuvant, resulting in anti-Hgp44 and anti-PorT antisera, respectively. Animal care and experimental procedures were conducted in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee. Preparation of anti-HbR antiserum has been described previously (20Nakayama K. Ratnayake D.B. Tsukuba T. Kadowaki T. Yamamoto K. Fujimura S. Mol. Microbiol. 1998; 27: 51-61Crossref PubMed Scopus (102) Google Scholar). Mass Spectrometry—In-gel digestion was performed as described previously (37Mortz E. Krogh T.N. Vorum H. Gorg A. Proteomics. 2001; 1: 1359-1363Crossref PubMed Scopus (461) Google Scholar). For MALDI-TOF analysis, digests were acidified with 1% trifluoroacetic acid and analyzed using the α-cyano-4-hydroxycinnamic acid thin layer technique on a 600-μm anchorchip target (Ultraflex TOF/TOF Bruker Daltonics, Bremen, Germany). For LC-MS analysis, acidified digests were preconcentrated and desalted on a C18 Pepmap precolumn and separated on a 75-μm C18 Pepmap column using an Ultimate nanoLC system (LC Packings, Amsterdam) and analyzed on-line by Ion Trap MS (Esquire HCT, Bruker Daltonics). MS/MS spectra were acquired automatically. Spheroplast Formation and Proteinase Treatment—-Spheroplast formation and proteinase treatment of P. gingivalis cells was essentially performed by the method described previously (38Delgado-Partin V.M. Dalbey R.E. J. Biol. Chem. 1998; 273: 9927-9934Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). After being suspended in 50 mm Tris acetate buffer (pH 7.8) containing 0.75 m sucrose, P. gingivalis cells were treated with lysozyme (final concentration, 0.1 mg/ml) on ice for 2 min. Conversion to spheroplasts was performed by slowly diluting the cell suspension over a period of 10 min with 2 volumes of cold 1.5 mm EDTA. After centrifugation at 10,000 × g for 10 min, the resulting precipitates were gently resuspended in 50 mm Tris acetate buffer (pH 7.8) containing 0.25 m sucrose and 10 mm MgSO4 (spheroplasts). The supernatants were used as the periplasm fraction and the proteins in this fraction were precipitated with trichloroacetic acid, and subjected to SDS-PAGE and immunoblot analysis. Formation of spheroplasts was examined by phase contrast microscopy. Spheroplasts were treated on ice with proteinase K (final concentration 1 mg/ml) in the presence or absence of 2% Triton X-100 for 1 h. After quenching proteinase K with phenylmethylsulfonyl fluoride (final concentration, 5 mm) for 5 min, the whole volume of the sample was mixed with 4 volumes of Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis. Registration of the Nucleotide Sequence Data—The GenBank™/EMBL/DDBJ accession number for the sequence reported in this study is AB016085. Isolation of a Non-pigmented Mutant of P. gingivalis by Transposon Mutagenesis, and Identification of a Gene Disrupted by the Transposon Insertion—Several non-pigmented clones were isolated among EmrP. gingivalis transconjugants after mating between Escherichia coli HB101 containing R751::Tn4351Ω4 and P. gingivalis 33277. One of the non-pigmented strains named KDP106 was further characterized in this study. Southern blot hybridization analysis revealed that the KDP106 chromosome contained a single Tn4351 insertion. A PvuII fragment (8 kb) of KDP106 chromosomal DNA that contained the inserted Tn4351 DNA was cloned by using the method of marker (Tcr on Tn4351 DNA) rescue. Sequencing of the flanking regions revealed that there was one ORF truncated by the transposon insertion. The ORF coding for 244 amino acids was designated porT (Fig. 1A). Construction of a PorT Mutant by Gene-directed Mutagenesis—To determine whether non-pigmentation of KDP106 was attributable to porT, we constructed a mutant with disruption of porT by gene-directed mutagenesis. We introduced the kan DNA block into the SalI region within porT and constructed a suicide vector plasmid containing the disrupted porT gene (pKD305). Introduction of pKD305 into P. gingivalis 33277 cells by electroporation produced a number of Emr transformants. Southern