Departments of Oral Infectious Diseases and Immunology1, Endodontology and Operative Dentistry2 and Pharmacology3, Faculty of Dental Science, Kyushu University, Fukuoka812-8582, Japan
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya464-8650, Japan4
Department of Medical Microbiology, Division of Molecular Pathology Infection and Immunity, St Bartholomews and the Royal London School of Medicine and Dentistry, London E1 2AA, UK5
Department of Microbiology, School of Dentistry, Nagasaki University, Nagasaki 852-8588, Japan6
Author for correspondence: Koji Nakayama. Tel: +81 95 849 7648. Fax: +81 95 849 7650. e-mail: knak{at}net.nagasaki-u.ac.jp
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ABSTRACT |
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Keywords: cell surface polysaccharide, cysteine proteinases, colonial pigmentation, haemagglutination, fimbrillin maturation
Abbreviations: HA, haemagglutinin; HbR, haemoglobin receptor protein; Kgp, Lys-gingipain; mt, membrane type; Rgp, Arg-gingipain; TLCK, N-p-tosyl-L-lysine chloromethyl ketone
The GenBank/EMBL/DDBJ accession number for the sequences reported in this paper is D64132.
a M.S. and D.B.R. contributed equally to this work.
b Present address: Department of Microbiology, School of Dentistry, Nagasaki University, Nagasaki 852-8588, Japan.
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INTRODUCTION |
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In early studies of P. gingivalis, pigment-less variants that were spontaneously isolated showed a weak ability to produce Rgp and Kgp, indicating that these proteinases or their genes might be responsible for accumulation of the µ-oxo dimers on the cell surfaces (McKee et al., 1988 ; Shah et al., 1989
). We found that the kgp mutant and the rgpA rgpB kgp triple mutant constructed by site-directed mutagenesis exhibited less and no pigmentation, respectively, which demonstrates the involvement of these gene products in haem storage on the cell surfaces (Shi et al., 1999
; Okamoto et al., 1998
).
Transposon mutagenesis has been applied to isolation of pigment-less mutants of P. gingivalis by several researchers (Genco et al., 1995a , b
; Chen et al., 2000
; Simpson et al., 1999
). Chen et al. (2000)
isolated nonpigmented mutants that had the transposon Tn4351 DNA within kgp. In addition, Simpson et al. (1999)
found that a nonpigmented 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 nonpigmentation have been found (Chen et al., 2000
; Abaibou et al., 2001
). Chen et al. (2000)
found that Tn4351 was inserted into a putative glucosyl (rhamnosyl) transferase-encoding gene in several nonpigmented mutants and Abaibou et al. (2001)
found that the gene vimA located downstream of recA is responsible for pigmentation.
In this study, we isolated a nonpigmented mutant that had a transposon insertion within the novel gene porR. The porR gene had homology with genes encoding transaminase involved in biosynthesis of sugar portions of LPSs and aminoglycosides. A porR mutant constructed by targeted mutagenesis with a suicide/integration plasmid also exhibited nonpigmentation. We found that the mutant showed a defect of polysaccharide biosynthesis and altered distribution of Rgp, Kgp, HA and HbR proteins in P. gingivalis cell fractions.
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METHODS |
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Gene-directed mutagenesis using a derivative of the suicide vector plasmid pKDCMZ (Nakayama, 1994 ) was essentially the same as described above except that E. coli DH5 harbouring R751 and pKD282, a derivative of pKDCMZ, was used as the donor strain.
Plasmid construction.
A PstI DNA fragment (6·8 kb) containing Tn4351 DNA of the chromosome of KDP105, a nonpigmented mutant isolated in this study, was cloned into a unique PstI site of pUC18, resulting in pKD267. The largest EcoRI fragment of pKD267 was self-ligated to yield pKD278 that contained one flanking region adhering to the transposition site of Tn4351. The NruI region (137 bp) of pKD278 was replaced by a kanamycin-resistance gene cartridge (1·3 kb) of pUC4K, resulting in pKD281. Then, an EcoRIPstI fragment (2·0 kb) of pKD281 was inserted into a unique SmaI site of suicide vector plasmid pKDCMZ, giving rise to pKD282.
Preparation of P. gingivalis cell fractions.
P. gingivalis cultures in 40 ml enriched BHI broth at various growth phases were centrifuged at 10000 g at 4 °C for 30 min. The supernatants of the cultures after the centrifugation were then subjected to ultracentrifugation at 100000 g at 4 °C for 2 h to separate into the fractions of culture supernatants and vesicles. The bacterial cell pellets were dissolved in 5 ml 10 mM Tris/HCl (pH 7·5) containing N-p-tosyl-L-lysine chloromethyl ketone (TLCK), leupeptin and EDTA at 0·1, 1 and 5 mM, respectively and disrupted by sonication to yield the crude cell extracts. The crude cell extracts were centrifuged at 10000 g for 1 h at 4 °C. The supernatants were saved and ultracentrifuged at 10000 g for 2 h at 4 °C, resulting in the membrane fraction. The vesicle and membrane fractions were directly dissolved in Laemmli buffer (Laemmli, 1970
) and subjected to SDS-PAGE (10% gel). The cell extracts and culture supernatants were diluted or concentrated, and mixed with the Laemmli buffer.
Enzymic assays.
Lys-X and Arg-X specific cysteine proteinase activities were determined by use of the synthetic substrates N-p-tosyl-Gly-Pro-Lys-p-nitroanilide (GPKpNA; Sigma) and N--benzoyl-DL-Arg-p-nitroanilide (BApNA; Sigma), respectively. In brief, various volumes of the cell lysates and supernatants of the culture were added to a reaction mixture (1 ml) containing 0·25 mM GPKpNA, 5 mM L-cysteine and 20 mM phosphate buffer (pH 7·5) for Kgp, and a reaction mixture (1 ml) containing 0·5 mM BApNA, 10 mM L-cysteine, 10 mM CaCl2, 100 mM Tris/HCl (pH 8·0) for Rgp. The reaction mixtures were incubated at 40 °C for Kgp and 30 °C for Rgp. After samples were added, the A405 was continuously measured on a spectrophotometer. Proteinase activities in cell extracts and culture supernatants were determined by increase in absorbance per min per ml culture.
Northern blot analysis.
Total RNA was extracted from P. gingivalis cells grown to mid-exponential phase (OD600 0·3) using an RNA purification kit (Qiagen RNeasy). Five microgrammes of RNA were electrophoresed in 1·2% agarose gel and then transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) according to the method described by Sambrook et al. (1989) . The antisense mRNA probes specific for the 0·5 kb BstXI (Tyr325)SphI (Met482) region of rgpB and the 0·5 kb AccI (Thr346)EcoRI (Gln510) region of kgp were constructed by using the pSPUTK plasmid (Stratagene). The RNA probes were labelled with digoxigenin using the DIG RNA labelling kit (Roche). Northern blot hybridization and detection were carried out according to the manufacturers recommendation.
RT-PCR.
This was done essentially according to the method of Matsuo et al. (1995) . Oligonucleotides 5'-ATGCCTACTTCCATTTCCCA-3' and 5'-TTGTAAAGACGGTTCGAATGTT-3' were used for detection of porR expression.
Gel electrophoresis and immunoblot analysis.
SDS-PAGE was performed essentially according to the method of Laemmli (1970) except that the sample buffer contained TLCK, leupeptin and EDTA at 0·1, 1 and 5 mM, respectively. For immunoblotting, proteins on SDS gels were electrophoretically transferred to nitrocellulose membranes using a semi-dry blotting system (Pharmacia). The blotted membranes were immunostained with anti-Rgp/Kgp antiserum (Kadowaki et al., 1998
), anti-HbR antiserum (Nakayama et al., 1998
), anti-fimbrillin antiserum (Nakayama et al., 1996
), mAb 61BG1.3 (Shi et al., 1999
) or mAb 1B5 (Curtis et al., 1999
), and signals were detected using an ECL detection system (Pharmacia).
Purification of cell surface polysaccharide.
This was done by the phenol/water method (Westphal & Jann, 1965 ). Visualization of the cell surface polysaccharide preparation on SDS-polyacrylamide gels was done according to Tsai & Frasch (1982)
.
Haemagglutination assay.
Cultures (24 h) of P. gingivalis strains in enriched BHI broth were centrifuged, washed with PBS and resuspended in PBS. The bacterial suspensions were then diluted in a two-fold series with PBS. A 100 ml aliquot of each dilution was mixed with an equal volume of sheep erythrocyte suspension (2·5% in PBS) and incubated in a round-bottom microtitre plate at room temperature for 3 h. The haemagglutination titre was determined as the last dilution exhibiting full agglutination.
Other methods.
Electrotransformation and Southern blotting were done as described previously (Nakayama et al., 1995 ).
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RESULTS |
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Construction of a porR mutant by gene-directed mutagenesis
To determine whether nonpigmentation of KDP105 was attributable to the disrupted ORF, we constructed a mutant with disruption of the ORF. We introduced the Kmr gene cartridge into the NruI region within the ORF and constructed a suicide vector plasmid containing the disruption (pKD282). Introduction of pKD282 into P. gingivalis ATCC 33277 by mobilization produced a number of Emr transconjugants. Southern blot hybridization analysis with chromosomal DNA of 10 Emr transconjugants revealed that the chromosomal DNA from all of the transconjugants examined possessed full-length plasmid pKD282 DNA inserted at the ORF region. The transconjugants were classified into three types with respect to the location of the Kmr gene cartridge (Fig. 1). Of these, the transconjugants with chromosomal structures I and II could be generated by reciprocal recombination with a single crossover event between the homologous DNA regions of the chromosome and the plasmid. However, the generation of structure III would require non-reciprocal recombination. This type of transconjugant was also obtained in our previous studies (Nakayama, 1994
, 1997
). Representative transconjugants exhibiting structures II and III were designated KDP108 and KDP107, respectively. Colonial pigmentation was determined by anaerobic growth of these strains on TS plates containing defibrinated and laked sheep blood. KDP107 showed no pigmentation, while KDP108 showed black pigmentation. This result demonstrated that the ORF was responsible for colonial pigmentation of P. gingivalis since both strains had the same chromosomal structure except for the ORF (Fig. 1
). The 5' portion of the ORF was cloned from the chromosome of KDP107 by the method of marker (Kmr) rescue. Combined with the nucleotide sequence of the 3' portion of the ORF, sequencing of this 5' region revealed the nucleotide sequence of the whole ORF (Fig. 1
). We named the ORF porR (regulation of porphyrin accumulation on the cell surface). We determined the porR expression of KDP107 and KDP108 by RT-PCR, with the result that KDP107 showed no expression of porR, while KDP108 did show expression (data not shown).
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(3) Immunoblot analyses using anti-Rgp/Kgp antiserum, anti-HA mAb and anti-HbR antiserum. Membrane fractions of the porR and porR+ strains grown in enriched BHI broth for 13, 17 and 21 h were subjected to SDS-PAGE and immunoblot analysis using anti-Rgp/Kgp antiserum (Fig. 5). The porR mutant had much smaller amounts of 50 kDa Rgp (RgpA and RgpB), and 55 kDa Kgp than the porR+ strain. Moreover, membrane-type Rgp (mt-RgpA and mt-RgpB) which was observed in the membrane fraction of the porR+ strain as diffuse heterogeneous bands of 7090 kDa, was not seen in the the porR mutant. Cell extracts, vesicle fractions and culture supernatants of the porR mutant grown in enriched BHI broth for 48 h were subjected to SDS-PAGE and immunoblot analysis (Fig. 6
). In immunoblot analysis using mAb 61BG1.3, which reacts with HA domains (HGP44 and HGP17), HA proteins with low molecular masses were observed in the culture supernatant of the porR mutant, whereas its cell extracts showed only immunoreacted protein bands with high molecular masses. On the other hand, the mature (low molecular mass) forms of HA proteins were seen in the cell extracts and culture supernatants of the wild-type strains. The vesicle fractions showed the same protein profiles as those of the cell extracts in both of the porR and porR+ strains (data not shown). In the immunoblot analysis using anti-HbR antiserum, HbR was present in all the fractions of the wild-type strains, whereas the porR mutant showed HbR not in the cell extracts or vesicle fractions but in the culture supernatants.
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DISCUSSION |
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Chen et al. (2000) isolated nonpigmented mutants that had a transposon insertion at a gene with homology to a glycosyl (rhamnosyl) transferase, an enzyme involved in the synthesis of O-antigen side chains of LPS. The mutants also showed reduced levels of Rgp and haemagglutinin activities. Interestingly, the rhamnosyl transferase gene was located approximately 1·5 kb upstream of porR. Although there has been no direct evidence to show the relationship with respect to gene expression between porR and the rhamnosyl transferase gene, these findings imply that both of the putative rhamnosyl transferase gene and porR may be involved in the same biosynthesis pathway.
Very recently, another nonpigmented mutant has been found in the study concerning P. gingivalis recA. A mutation of the vimA gene located downstream of recA was responsible for deficiency in pigmentation and haemagglutination of this mutant (Abaibou et al., 2001 ). Moreover, Rgp and Kgp were mostly found in particle-free medium of the vimA mutant. Cell suspension and vesicles of the mutant showed very little gingipain activity. These properties of the vimA mutant are very similar to those of the porR mutant. We constructed a vimA mutant using gene replacement with vimA::Emr and found that the vimA mutant produced no mAb 1B5-reactive substances (unpublished data). These findings indicate that vimA may also be involved in biosynthesis of the saccharide portions of mt-Rgp and cell surface polysaccharide.
Rgp and Kgp were produced but not retained on the cell surfaces of the porR mutant and most of the gingipains were found in the culture supernatants. Moreover, the mature forms of HA and HbR proteins were also released into the culture supernatants, leading to no haemagglutination of the mutant cells. These results indicate that the porR mutant is incapable of retaining those mature proteins derived from rgp and kgp genes on the cell surfaces. A defect of the cell surface polysaccharide in the mutant may cause this incapability since levels of major outer-membrane proteins are drastically decreased in Salmonella typhimurium, E. coli and Shigella flexneri mutants synthesizing very defective LPS such as the deep rough mutants (Koplow & Goldfine, 1974 ; Ames et al., 1974
; Sandlin et al., 1995
). Potempa et al. (1995)
found that distribution of Rgp and Kgp activities in P. gingivalis cell fractions are different among P. gingivalis strains. Strain H66 produces Rgp and Kgp mostly in particle-free culture supernatants, whereas strains ATCC 33277 and W50 produce them in all the fractions (particle-free culture supernatants, vesicles, membrane fractions and membrane-free cell extracts). The difference in Rgp and Kgp distribution among P. gingivalis strains might be related to the degree of production or maturation of cell surface polysaccharide.
In our previous studies (Kadowaki et al., 1998 ; Nakayama et al., 1996
) we found that FimA fimbrillin remained in a precursor form in the Rgp-null mutant and that prefimbrillin expressed in E. coli was converted to the mature fimbrillin in vitro when incubated with purified Rgp, but its conversion was suppressed by potent Rgp inhibitors. The porR mutant cells in early exponential phase produced a precursor form of FimA fimbrillin and after prolonged incubation, the precursor was converted into a mature form of FimA fimbrillin. The molecular mass of the FimA precursor was 43·5 kDa, 0·5 kDa larger than that of the mature FimA fimbrillin. This indicates that the precursor has about five additional residues compared to the mature FimA, which is consistent with our recent finding that the precursor form of FimA accumulated on the cell surface of the rgpA rgpB kgp mutant had six additional residues (TSNSNR) at the amino terminus compared to the mature FimA (unpublished data). Fimbrillin maturation of the porR mutant appears to be synchronized with the increase of Rgp/Kgp activity. The present results strongly suggest that conversion of prefimbrillin to mature fimbrillin in vivo is dependent on Rgp/Kgp activity.
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ACKNOWLEDGEMENTS |
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Received 29 August 2001;
revised 20 November 2001;
accepted 21 November 2001.