1 Division of Microbiology and Oral Infection, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan
2 Division of Oral Molecular Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan
Correspondence
Koji Nakayama
knak{at}net.nagasaki-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of the ustA gene region of P. gingivalis ATCC 33277 is AB188568.
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INTRODUCTION |
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Porphyromonas gingivalis is a Gram-negative anaerobic bacterium belonging to the phylum Bacteroidetes (Boone et al., 2001). This bacterium is one of the organisms that are strongly associated with chronic adult periodontitis and expresses numerous potential virulence factors, such as fimbriae, haemagglutinins, lipopolysaccharides and various proteases that are capable of hydrolysing collagen, immunoglobulins, iron-binding proteins and complement factors (Holt et al., 1999
; Lamont & Jenkinson, 1998
). To facilitate adaptation to life within the oral cavity, P. gingivalis must be capable of sensing and responding to the prevailing environmental conditions, including variations in temperature, oxygen tension, pH, nutrient availability and the presence of other bacterial or host cells. Lu & McBride (1994)
found that P. gingivalis homologues of DnaK and GroEL were upregulated when cells were shifted from 37 to 42 °C. These proteins were also induced by treatment with ethanol, but not by oxidative stress or change in pH. In addition, Shelburne et al. (2002)
reported that P. gingivalis cells stressed in vitro by a 5 °C temperature increase showed a rapid rise in the mRNA associated with genes encoding the molecular chaperones (htpG, dnaK, groEL), superoxide dismutase (sod) and gingipain (rgpA). Nevertheless, there is relatively little information on the mechanisms that may operate in this bacterium in response to entrance into the stationary growth phase.
In this study, we describe the identification of a gene whose product is characterized as a novel P. gingivalis protein with a molecular mass of 9 kDa and an isoelectric point of 4·5 that is upregulated in stationary phase (UstA) by using two-dimensional (2D) gel electrophoresis. Chromosomal mutants carrying a disruption of the ustA gene are constructed and analysed to gain insights to the physiological role of UstA.
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METHODS |
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Oxidative stress conditions.
Cells of P. gingivalis were grown anaerobically at 37 °C in enriched BHI broth. Exponential-phase (15 h after inoculation) or stationary-phase (48 h after inoculation) cultures were divided into two portions (40 ml each): one portion was left under anaerobic conditions and the other was incubated aerobically for 20 or 120 min with vigorous shaking at 150 r.p.m.
2D gel electrophoresis.
P. gingivalis cells were harvested and treated with 10 % trichloroacetic acid. The resulting precipitates were washed several times with acetone and dried. Cells were then suspended in a lysing buffer (8 M urea, 4 % CHAPS, 20 mM DTT and 40 mM Tris base). The lysates were sonicated and centrifuged at 22 000 g for 5 min. After centrifugation, the supernatant was subjected to 2D gel analysis. 2D gel electrophoresis was performed by using the MultiphorII system (Amersham Pharmacia) according to the manufacturer's instructions. Briefly, 18 cm dehydrated isoelectric focusing strips with an immobilized pH gradient between pH 4 and 7 were rehydrated overnight in a rehydration solution [8 M urea, 20 mM DTT, 4 % CHAPS and 0·00001 % bromophenol blue (BPB) in 10 ml distilled water]. Then, each protein preparation at the same concentration was loaded on rehydrated gel strips and proteins were electrofocused at 20 °C in four stages (500 V, 2 mA, 5 W for 1 min; 500 V, 2 mA, 5 W for 1 h; 3500 V, 2 mA, 5 W for 1·5 h; and 3500 V, 2 mA, 5 W for 6 h). Focused gel strips were equilibrated for 10 min in equilibration solution A (50 mM Tris/HCl, pH 8·8, 30 % glycerol, 6 M urea, 2 % SDS, 40 mM DTT) and for another 10 min in 10 ml equilibration solution B (50 mM Tris/HCl, pH 8·8, 30 % glycerol, 6 M urea, 2 % SDS, 0·00001 % BPB, 135 mM iodoacetamide). For the second dimension electrophoresis, we used precast polyacrylamide gels with a linear polyacrylamide gradient from 12 to 14 % with the appropriate precast buffer strips (Amersham Pharmacia). Gels were run at 1000 V, 20 mA and 40 W at 15 °C for 45 min, after which the first dimension strip gels were removed. The gel was then run at 1000 V, 40 mA and 40 W at 15 °C for 180 min. Proteins in the gels were stained with Coomassie brilliant blue (CBB) or electrophoretically transferred onto a PVDF membrane (Millipore) by the method of Matsudaira (1987).
Protein and DNA sequencing.
After transfer of proteins onto the PVDF membrane, protein spots were cut from the membrane and subjected to protein sequencing using a model 470A gas-phase protein sequencer (Applied Biosystems). DNA sequencing was performed using plasmid templates and a dideoxy sequencing kit (Thermal cycler sequencing kit; Amersham Pharmacia) with a Long Reader Sequencer 4200 (Li-Cor).
Primer extension analysis.
Total RNA was extracted from P. gingivalis cells grown to the stationary phase (OD600=0·8) by using TRIzol reagent (Invitrogen). A 30-mer oligonucleotide (PE1) corresponding to the DNA region starting with a C residue 91 nucleotides downstream of the ustA initiation codon was radiolabelled by T4 polynucleotide kinase (Takara) with [-32P]ATP (Amersham Pharmacia). Oligonucleotides including PE1 used in this study were purchased from Invitrogen and are listed in Table 2
. RNA samples (50 µg) were incubated with 0·5 pmol radiolabelled oligonucleotide primer at 60 °C for 60 min and then at 37 °C for 90 min. After addition of dNTPs (0·5 mM each) and Superscript II RNase H reverse transcriptase (200 U; Gibco Laboratories), the mixtures were incubated at 42 °C for 1 h. The resulting products were loaded on a 5 % Long Ranger (FMC) polyacrylamideurea gel and electrophoresed together with DNA samples that were obtained from a sequencing reaction of the corresponding double-stranded DNA with the same oligonucleotide primer.
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Subcellular fractionation.
P. gingivalis cells were harvested from 3000 ml full-grown culture by centrifugation at 10 000 g at 4 °C for 30 min and resuspended in 100 ml PBS containing 0·1 mM TLCK, 0·1 mM leupeptin and 0·5 mM EDTA. The cells were disrupted in a French pressure cell at 100 MPa. Unbroken cells were removed by centrifugation at 1000 g for 10 min and the supernatant was subjected to ultracentrifugation at 100 000 g for 60 min. The precipitates were treated with 1 % Triton X-100 in PBS containing 20 mM MgCl2 at 20 °C for 30 min. The inner and outer membrane fractions were recovered as a supernatant and a pellet, respectively, by ultracentrifugation at 100 000 g at 4 °C for 60 min (Murakami et al., 2002). Proteins were subjected to Tris-Tricine SDS-PAGE and transferred to PVDF membranes and immunoreacted with anti-UstA antiserum.
Preparation of anti-UstA antiserum.
A peptide derived from the amino acid sequence of UstA (G62S77) with an N-terminal cysteine residue, CGIVELDDTTILERALS, which was conjugated to keyhole limpet haemocyanin, was purchased from Sigma Genosys. The conjugated peptide (250 µg) was mixed with Freund's complete adjuvant and injected subcutaneously into a rabbit (Japan White) with two booster shots of a mixture of the conjugated peptide and Freund's incomplete adjuvant. 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.
Construction of P. gingivalis ustA : : erm mutants.
The chromosomal DNA region including the ustA gene (3·3 kb) was PCR-amplified from the chromosomal DNA of P. gingivalis ATCC 33277 (referred to as strain 33277) using Advantage 2 DNA polymerase (BD Biosciences) with the oligonucleotides USTAFOR and USTAREV (Table 2) as upstream and downstream primers, respectively. The amplified DNA fragment was cloned into a T-vector (pGEM-T Easy; Promega), resulting in pKD801. The ermFermAM DNA cassette of pKD355 (Ueshima et al., 2003
) was inserted into the BsiWI site within ustA of pKD801 to yield pKD802. pKD802 plasmid DNA was linearized by NotI digestion and introduced into cells of P. gingivalis 33277 and KDP143 (oxyR : : tetQ) by electroporation as described previously (Nakayama et al., 1995
), resulting in strains KDP301 (ustA : : ermF ermAM) and KDP306 (ustA : : ermF ermAM oxyR : : tetQ), respectively.
To construct the ustA+ complemented mutant, the whole ustA gene region with its upstream and downstream flanking regions (0·6 kb) was PCR-amplified from the chromosomal DNA of P. gingivalis 33277 using Pyrobest DNA polymerase (Takara) with the oligonucleotides CP1 and CP2 (Table 2) as upstream and downstream primers, respectively. The amplified DNA fragment (559 bp) was cloned into pCR-Blunt II-TOPO (Invitrogen) according to the manufacturer's recommendation. The resulting plasmid pKD803 was subjected to DNA sequencing. In addition, the tetQ DNA cassette of pKD375 (Shi et al., 1999
) was inserted into the BamHI site of pKD703, a plasmid containing both flanking DNA regions of P. gingivalis fimA (Shoji et al., 2004
), to yield pKD713. The whole ustA region (0·6 kb) of pKD803 obtained by EcoRI digestion was inserted into the EcoRI site of pKD713 to yield pKD804. Plasmids pKD713 and pKD804 were linearized by BssHII digestion and introduced into KDP301 (ustA : : ermF ermAM) by electroporation, resulting in KDP302 (ustA : : ermF ermAM fimA : : tetQ) and KDP303 (ustA : : ermF ermAM fimA : : tetQ ustA+), respectively, after 7 days incubation on enriched TS agar containing 1·0 µg tetracycline ml1. Correct gene replacement of these strains that had been generated by double crossover recombination events was verified by PCR and Southern blot analysis.
Construction of P. gingivalis strains containing the sod''lacZ protein fusion gene.
To construct an sod''lacZ protein fusion gene, a DNA fragment comprising the 5'-terminal region of sod and its upstream region was PCR-amplified from the chromosomal DNA of P. gingivalis 33277 with primers EX1 and EX2 (Table 2). Primer EX1 can hybridize to the chromosomal DNA 0·92 kb upstream of the initiation codon of sod and generate a BamHI site at one end of the PCR product, while primer EX2 can hybridize to the chromosomal DNA within the sod gene and generate a BamHI site at the other end of the PCR product. The amplified DNA fragment was cloned into pGEM-T Easy, sequenced, excised by digestion with BamHI and ligated to BamHI-digested DNA of the lacZ reporter suicide/integration plasmid pKD393 (Ueshima et al., 2003
). The correct orientation of the insert DNA was confirmed by sequencing. The resulting plasmid pKD398 DNA encoded a recombinant
-galactosidase fused to the N-terminal 17 amino acids of P. gingivalis Sod. Circular plasmid DNA of pKD398 was then introduced into cells of P. gingivalis 33277 by electroporation to yield KDP151 (sod''lacZ). Moreover, the tetQ DNA cassette of pKD375 was inserted into the BsiWI site within ustA of pKD801 to yield pKD805. KDP151 was transformed to tetracycline resistance by electrotransformation with NotI-linearized pKD805 (ustA : : tetQ), resulting in KDP305 (sod''lacZ ustA : : tetQ). Correct DNA integration or replacement in KDP151 and KDP305 was confirmed by Southern hybridization.
DNA probes and Southern blot hybridization.
A DNA fragment (523 bp) comprising the 3'-terminal region of ustA and its downstream region was PCR-amplified from the chromosomal DNA of P. gingivalis 33277 with the oligonucleotide primers SH1 and SH2 (Table 2). The ermFermAM DNA cassette (2·1 kb) obtained from pKD355 by BamHI and BglII double digestion was labelled with the AlkPhos Direct system for chemiluminescence (Amersham Pharmacia). Southern blotting was performed by using a nylon membrane and developed with CDP-star detection reagent (Amersham Pharmacia).
Assays for superoxide dismutase (SOD) and -galactosidase activities.
P. gingivalis cells were harvested, suspended in 0·1 M phosphate buffer (pH 7·5) containing 0·1 mM TLCK and 0·1 mM leupeptin and sonicated. Extracts were assayed for SOD activity by the cytochrome cxanthine oxidase method (McCord & Fridovich, 1969). For
-galactosidase assays, P. gingivalis cells were suspended in buffer Z (Miller, 1972
) and subjected to the
-galactosidase assays described in Miller (1972)
. All assays were performed in duplicate and repeated at least three times.
Agar diffusion assay.
P. gingivalis cells were grown anaerobically in enriched BHI broth and spread on enriched TS agar plates and a sterile disc containing 3 µl of solutions containing 6 % H2O2, 15 % H2O2, 7 % t-BOOH, 14 % t-BOOH, 10 % CM-OOH, 20 % CM-OOH, 200 mM diamide, 500 mM diamide, 0·5 mg metronidazole ml1, 1·0 mg metronidazole ml1 or 1·0 mg mitomycin C ml1 was placed at the centre of each plate and incubated anaerobically at 37 °C for 4 days.
General procedures.
Unless otherwise stated, standard procedures were used for the preparation and handling of DNA and RNA (Sambrook et al., 1989).
Statistical analysis.
Student's t test was used to compare differences in inhibitory zone width among bacterial strains by using StatView J4.5 software (Abacus Concepts).
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RESULTS |
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DISCUSSION |
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This study demonstrates that a 9 kDa protein of P. gingivalis, the gene of which is located upstream of uspA, is upregulated in stationary phase, and we propose to call it UstA. UstA may be involved in oxidative stress responses, since expression of redox proteins such as Sod, Tpx and Trx is increased in the ustA mutant. UstA homologues were found in only the genus Bacteroides in addition to the genus Porphyromonas, implying that UstA may be present in rather restricted bacterial groups and may regulate oxidative stress responses in response to environmental conditions.
In these bacterial species, P. gingivalis, B. fragilis and B. thetaiotaomicron, the usp gene is located downstream of ustA in the same orientation (Nelson et al., 2003; Tang et al., 1999
; Xu et al., 2003
). The production of Usp in various bacteria was found to be stimulated by a large variety of conditions, such as stationary phase, starvation of carbon, nitrogen, phosphate, sulphate and amino acids and exposure to heat, oxidants, metals, uncouplers, polymyxin, cycloserine, ethanol, antibiotics and other stimulants (Gustavsson et al., 2002
; Kvint et al., 2003
). Primer extension and Northern blot hybridization analyses revealed that ustA was transcribed in a monocistronic fashion. We have not found so far that expression of these two genes is co-ordinately regulated; however, they may be related to each other with respect to stress responses. Further studies are contemplated to explore the interaction of UstA with Usp.
The ustA mutant was more resistant to diamide than the wild-type parent strain, which can be explained by overexpression of Tpx and Trx in the ustA mutant, since Tpx interacting with Trx functions as a thiol-specific antioxidant (Cha et al., 1995, 1996
). In contrast, sensitivities of the ustA single mutant to H2O2, t-BOOH, CM-OOH, mitomycin C and metronidazole were not significantly different from those of the wild-type parent. There are several possible explanations as to why the ustA mutant is not more resistant to peroxides despite the fact that Sod, Tpx and Trx are significantly upregulated in the mutant. Firstly, other proteins that protect the cells from peroxide stress may be downregulated in the ustA mutant. Secondly, the UstA protein itself may contribute to protection against peroxide stress. Further work is needed to clarify this point. On the other hand, the oxyR mutant showed hypersensitivity to all of the chemicals compared with the wild-type parent. In E. coli, the redox-sensitive transcription activator OxyR regulates a number of genes, many of which have clear antioxidant roles (Storz et al., 1990
; Storz & Zheng, 2000
). In B. fragilis, katB, dps, ahpC, tpx and rbpA have been found to be under OxyR control (Rocha et al., 2000
; Herren et al., 2003
). Hypersensitivity of the P. gingivalis oxyR mutant to oxidants and DNA-damaging agents suggests that OxyR may regulate expression of antioxidants in this bacterium also. Interestingly, the ustA oxyR double mutant was markedly more resistant to metronidazole than the oxyR mutant. Metronidazole is a prodrug, which requires reduction of the nitro group for bactericidal activity (Edwards, 1993
). Under anaerobic conditions, metronidazole is reduced by microbial nitroreductases to a cytotoxic nitro radical, which interacts with DNA, resulting in damage characterized by helix destabilization and strand breakage (Lindmark & Muller, 1976
; Moreno et al., 1983
; Goodwin et al., 1998
; Jeong et al., 2001
; Rasoloson et al., 2002
). The ustA mutation may compensate for the defect of the oxyR mutant by downregulating expression of putative metronidazole-activating nitroreductases or upregulating expression of DNA-repair enzymes. Suppression of the oxyR phenotypes by the ustA mutation was also seen in sensitivity to diamide and mitomycin C. The suppressive effects of the ustA mutation might be partially explained by the increase of Sod, Tpx or Trx in the ustA mutant, but participation of other redox proteins or DNA-repair enzymes might not be ruled out.
2D gel analysis revealed that expression of Sod, Tpx and Trx was increased in the ustA mutant. Using the sod''lacZ protein fusion strain, we found that the -galactosidase activity in the ustA mutant was about twice that of the wild-type under anaerobic conditions and was 2·4-fold higher 2 h after exposure to atmospheric oxygen, consistent with the result that the SOD activity of the ustA mutant was higher than those of the wild-type parent and the ustA+ complemented strain; however, the increase of
-galactosidase activity cannot account for the whole increase in SOD activity. This apparent difference might be explained by possible instability of the chimeric
-galactosidase encoded by the sod''lacZ protein fusion gene and/or duplication of the sod promoter DNA region in the sod''lacZ protein fusion strain. The mechanisms of effects of UstA on expression of Tpx and Trx also remain to be determined.
In conclusion, we found a novel 9 kDa protein named UstA in P. gingivalis that was induced in stationary phase or oxidative conditions. Sod, Tpx and Trx were upregulated in the ustA insertional mutant. The ustA mutation conferred resistance to diamide on P. gingivalis wild-type cells and suppressed hypersensitivities of the oxyR mutant to diamide, metronidazole and mitomycin C. These results suggest that UstA plays a significant role in oxidative stress responses in P. gingivalis.
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ACKNOWLEDGEMENTS |
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Received 26 August 2004;
revised 5 November 2004;
accepted 13 December 2004.
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