J Med Microbiol 57 (2008), 1411-1419; DOI: 10.1099/jmm.0.2008/001677-0
© 2008 Society for General Microbiology
ISSN 0022-2615
Identification and molecular analysis of βC–S lyase producing hydrogen sulfide in Streptococcus intermedius
Shuntaro Ito1,2,
Hideaki Nagamune3,
Haruki Tamura1 and
Yasuo Yoshida1
1 Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan
2 Department of Periodontology, Iwate Medical University School of Dentistry, Morioka, Japan
3 Department of Biological Science and Technology, Life System, Institute of Technology and Science, University of Tokushima Graduate School, Tokushima, Japan
Correspondence
Yasuo Yoshida
yasuoy{at}iwate-med.ac.jp
Received March 31, 2008
Accepted August 5, 2008
Hydrogen sulfide (H2S) is a toxic gas that induces the modification and release of haemoglobin in erythrocytes; however, it also functions in methionine biosynthesis in bacteria. βC–S lyase, encoded by the lcd gene, is responsible for bacterial H2S production through the cleavage of L-cysteine. In this study, 26 of 29 crude extracts from reference and clinical strains of Streptococcus intermedius produced H2S from L-cysteine. The capacities in those strains were not higher than those in strains of the other anginosus group of streptococci, Streptococcus anginosus and Streptococcus constellatus, but were much greater than those in strains of Streptococcus gordonii, which is known to have an extremely low capacity for H2S production. Incubation of the remaining three extracts with L-cysteine did not result in H2S production. Sequence analysis revealed that the lcd genes from these three strains (S. intermedius strains ATCC 27335, IMU151 and IMU202) contained mutations or small deletions. H2S production in crude extracts prepared from S. intermedius ATCC 27335 was restored by repairing the lcd gene sequence in genomic DNA. The kinetic properties of the purified recombinant protein encoded by the repaired lcd gene were comparable to those of native proteins produced by H2S-producing strains, whereas the truncated protein produced by S. intermedius ATCC 27335 had no enzymic activity with L-cysteine or L-cystathionine. However, real-time PCR analysis indicated that the lcd gene in strains ATCC 27335, IMU151 and IMU202 is transcribed and regulated in a manner similar to that in the H2S-producing strain.
Abbreviations: PLP, pyridoxal 5'-phosphate; GST, glutathione S-transferase.
The GenBank/EMBL/DDBJ accession numbers for the lcd sequences of S. intermedius strains ATCC 27335, IMU202, IMU151, IMU105, IMU122, IMU201 and DP102 are AB381917, AB381918, AB381919, AB381920, AB381921, AB381922 and AB381923, respectively.
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INTRODUCTION
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Micro-organisms require sulfur for the production of cysteine and methionine, which are essential for protein biosynthesis. Sulfur is also a constituent of several other indispensable cellular components, including thiamine, biotin, lipoic acid and coenzyme A. In general, sulfur is assimilated via the sulfate pathway, which involves the reduction of sulfate (
) to sulfide (S2–) and the subsequent incorporation of S2– into cysteine. Cysteine serves as a sulfur donor in the biosynthesis of methionine through the formation of homocysteine (Fig. 1
). There are two alternative pathways for homocysteine biosynthesis: the trans-sulfuration pathway, in which cystathionine 
-synthase and cystathionine β-lyase transfer a sulfur atom from cysteine to homocysteine via cystathionine, a thioester intermediate, and the direct sulfhydrylation pathway, in which sulfur from hydrogen sulfide (H2S) is directly fixed with O-acetylhomoserine or O-succinylhomoserine by acylhomoserine sulfhydrylase (Fig. 1). Ultimately, homocysteine is converted to methionine by methionine synthase (Soda, 1987). Enteric bacteria, such as Escherichia coli, use the trans-sulfuration pathway (Smith, 1971), whereas organisms such as Saccharomyces cerevisiae (Thomas & Surdin-Kerjan, 1997), Rhizobium etli (Tate et al., 1999), Pseudomonas aeruginosa (Foglino et al., 1995) and Leptospira meyeri (Belfaiza et al., 1998) use the direct sulfhydrylation pathway.
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Fig. 1. Two pathways for the biosynthesis of methionine in bacteria. Genes encoding enzymes are indicated in parentheses. O-Acetyl-L-homoserine can be replaced by O-succinyl-L-homoserine.
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We previously reported that both of these pathways function in Streptococcus anginosus (Yoshida et al., 2003b). In the biosynthesis of homocysteine, the protein encoded by the cgs gene functions as a cystathionine -synthase in the trans-sulfuration pathway and as an acylhomoserine sulfhydrylase in the direct sulfhydrylation pathway (Fig. 1
). In contrast, the lcd gene, which is co-transcribed with cgs as an operon, encodes βC–S lyase, also known as cystathionine β-lyase. This pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyses the 
,β-elimination of L-cysteine and L-cystathionine to produce H2S and homocysteine, respectively (Yoshida et al., 2002). βC–S lyase shows a high H2S-producing capacity in S. anginosus and Streptococcus constellatus (Yoshida et al., 2008), both of which belong to the anginosus group streptococci with Streptococcus intermedius (Kawamura et al., 1999; Whiley et al., 1990; Whiley & Beighton, 1991), whereas homologues from other oral streptococci, such as Streptococcus gordonii, Streptococcus oralis, Streptococcus mutans, Streptococcus sobrinus and Streptococcus salivarius, are able to produce homocysteine from L-cystathionine, but are almost unable to form H2S from L-cysteine (Yoshida et al., 2003a). Hence, it appears that S. anginosus and S. constellatus use both the trans-sulfuration and direct sulfhydrylation pathways, whereas other streptococci that have been tested use only the former pathway. However, it is unclear whether the capacity to produce large amounts of H2S is also common to the remaining species of the anginosus group (e.g. S. intermedius).
For this reason, we conducted molecular and enzymic studies of βC–S lyase associated with production of H2S and homocysteine in the methionine biosynthetic pathway of S. intermedius. The lcd gene encoding βC–S lyase in S. intermedius strains and its products were characterized. The effects of lcd gene disruption, which were identified in the course of this study, were also characterized in three strains.
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METHODS
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Bacterial strains, culture conditions and genetic methods.
The strains used in this study are listed in Table 1. The clinical isolates, which are indicated by the prefix IMU, were collected from clinical specimens submitted for culture to the Central Clinical Laboratory, Iwate Medical University Hospital (Morioka, Japan). Each IMU strain was isolated from a specimen collected from a distinct patient. The strains were identified as S. intermedius by PCR as previously described (Takao et al., 2004). The source of other clinical isolates is described elsewhere (Whiley & Beighton, 1991; Nagamune et al., 2000). The streptococci were grown anaerobically in brain heart infusion (BHI; Difco) broth at 37 °C. When required, spectinomycin or erythromycin was used to supplement the media at 250 or 10 µg ml–1, respectively. E. coli strains DH5 (Invitrogen) and BL21 (Promega) were used for DNA manipulation and protein purification, respectively, and were grown aerobically at 37 °C in 2xYT broth or agar (Difco), which was supplemented with 20 µg chloramphenicol ml–1or 100 µg ampicillin ml–1 as required for maintenance of the plasmids.
Cloning and sequencing of the lcd genes.
PCR fragments (
1.3 kb) containing the lcd gene from the S. intermedius strains ATCC 27335, IMU105, IMU122, IMU201, IMU202, IMU151 and DP102 were amplified using primers specific for the flanking regions of lcd in S. anginosus and S. constellatus (Yoshida et al., 2008). Each amplified fragment was then cloned into pMCL210 (Nakano et al., 1995) and sequenced using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The sequences were assembled and analysed using Vector NTI software (Invitrogen).
Preparation of crude enzyme extracts.
Crude enzyme extracts were obtained as previously described (Yoshida et al., 2002). Briefly, each streptococcal strain was grown in BHI to an OD600 of approximately 1.0, which corresponded to late exponential phase for all the strains. The cells were then harvested from 200 ml of culture and washed three times with cold PBS (0.12 M NaCl, 0.01 M Na2HPO4 and 5 mM KH2PO4, pH 7.5). A 500 µl aliquot of the cell suspension was then transferred to a screw-cap microcentrifuge tube containing 0.5 g of 0.1–0.15 mm-diameter glass beads. After vortexing the cells with the beads ten times for 30 s at 1 min intervals, the supernatant was centrifuged. The protein concentration in the extracts was then determined using a protein assay reagent (Bio-Rad) with BSA as a standard. After the addition of an equal volume of 80 % (v/v) glycerol, the samples were stored at –20 °C.
Repair of the lcd pseudogene in S. intermedius ATCC 27335.
To obtain an S. intermedius ATCC 27335 mutant strain containing a repaired lcd gene, we initially constructed the mutant strain KO100, in which the entire region of the lcd pseudogene was replaced by a spectinomycin-resistance gene (spc). The intermediate mutant strain was produced to avoid unexpected recombination in the next step. The DNA fragment used for transformation was prepared by overlap extension PCR (Horton et al., 1989), as previously described (Yoshida et al., 2005). Briefly, each reaction mix contained KOD Hot Start DNA Polymerase (Toyobo), a template consisting of three overlapping PCR fragments (the upstream gene targeting sequence, the spc gene and the downstream gene targeting sequence) and primers complementary to the 5' end of the upstream gene targeting sequence and the 3' end of the downstream gene targeting sequence. Individual fragments were prepared by the amplification of spc from pDL278 (LeBlanc et al., 1992) and targeting sequences from S. intermedius ATCC 27335 genomic DNA using appropriately designed primers. Next, the spc gene in strain KO100 was replaced with the repaired lcd linked to the erythromycin-resistance cassette (ermAM). Transforming DNA was prepared by three rounds of overlap extension PCR: the first to link the upstream gene targeting sequence to the repaired lcd, the second to link ermAM to the downstream gene targeting sequence and the third to link the two constructs together. The primers used to amplify these fragments were designed to create complementarity between the constructs. The repaired lcd was also amplified by overlap extension PCR using two complementary primers with an inserted cytosine. The ermAM cassette and the gene targeting sequences were prepared from pKSerm2 (Lunsford, 1995) and S. intermedius ATCC 27335 genomic DNA, respectively.
The transformation of S. intermedius was performed as previously described (Lunsford, 1995) with minor modifications. Briefly, an overnight Todd–Hewitt broth (THB) culture was diluted 1 : 20 in THB containing 5 % (v/v) heat-inactivated horse serum (THB-HS). Following incubation for 2 h at 37 °C, the resulting culture was again diluted 1 : 20 in fresh THB-HS and incubated for 2 h at 37 °C to obtain early-exponential-phase competent cells. Transformation reactions containing 50 µl competent cells, 450 µl THB-HS, 2 µg transforming DNA and 50 ng competence-simulating peptides (Havarstein et al., 1997) were incubated for 2 h at 37 °C prior to plating on BHI agar containing 5 % (v/v) heat-inactivated horse serum and spectinomycin or erythromycin.
Purification of recombinant βC–S lyase.
Recombinant Lcd proteins from the S. intermedius strains ATCC 27335, IMU201 and KO101 were purified using the expression vector pGEX-6P-1 (GE Healthcare), as previously described (Yoshida et al., 2002). Briefly, each lcd gene was amplified by PCR using primers designed to incorporate a BamHI site at the 5' end and a SalI or XhoI site at the 3' end of each segment. Following amplification, the products were digested with the appropriate restriction enzymes and ligated into pGEX-6P-1, juxtaposing each lcd fragment downstream of the coding sequence for glutathione S-transferase (GST) and a PreScission protease (GE Healthcare) cleavage site. The recombinant E. coli clones were grown in 2xYT broth containing ampicillin to an OD600 of about 0.8, and IPTG was added to a final concentration of 0.5 mM. The cells were harvested 2 h after induction, washed with PBS, and then lysed by ultrasonication. After centrifugation at 15 000 g for 30 min at 4 °C, the lysates were incubated with glutathione-Sepharose 4B beads (GE Healthcare). After extensive washing, the beads were treated with PreScission protease to recover the purified protein. The protein concentration was determined using Bio-Rad protein assay reagent, and the purity of the samples was verified by SDS-PAGE.
Visualization of enzymic activity.
L-Cysteine desulfhydrase activity of crude enzyme extracts was visualized using non-denaturing polyacrylamide gels as previously described (Claesson et al., 1990). The samples were electrophoresed at 10 mA per gel at 4 °C for 2.5 h on 12.5 % (w/v) resolving (pH 8.8) and 3 % (w/v) stacking (pH 6.5) polyacrylamide gels. After electrophoresis, the gel was incubated in visualizing solution (100 mM triethanolamine.HCl, pH 7.6, 10 µM PLP, 0.5 mM bismuth trichloride, 10 mM EDTA and 5.0 mM L-cysteine) at 37 °C to allow detection of the enzyme's position.
Enzyme activity assay.
The level of activity of the βC–S lyases was examined by measuring the rate of formation of H2S or pyruvate. To estimate H2S production, a methylene blue formation assay was performed following the method of Schmidt (1987). Briefly, the reaction mixture contained the following reagents in a final volume of 200 µl: 40 mM potassium phosphate buffer (pH 7.6), 2.5 mM dithioerythritol, 10 µM PLP, 2.0 mM L-cysteine and 53.6 µg crude enzyme extract. After 10 min of incubation at 37 °C, the reaction was terminated by the addition of 20 µl solution I (20 mM N',N'-dimethyl-p-phenylenediamine dihydrochloride in 7.2 M HCl) and 20 µl solution II (30 mM FeCl3 in 1.2 M HCl). After incubation for 30 min at room temperature, methylene blue formation was examined spectrophotometrically at 670 nm using a molar absorption coefficient of 28.5x106 M–1 cm–1.
Pyruvate formation was measured as previously described (Soda, 1968). The assay was carried out in a reaction mixture (100 µl) containing 50 mM potassium phosphate buffer (pH 7.6), 1 nmol PLP, 170 ng (for L-cysteine) or 40 ng (for L-cystathionine) purified enzyme and various amounts of each substrate. After 2 min incubation at 37 °C, the reaction was terminated by the addition of 50 µl 4.5 % (v/v) trichloroacetic acid. The reaction mixture was then centrifuged and 100 µl of the supernatant was added to 300 µl 0.67 M sodium acetate (pH 5.2) containing 0.017 % (w/v) 3-methyl-2-benzothiazolinone hydrazone. After incubation at 50 °C for 30 min, the absorbance at 335 nm was determined. The amount of pyruvate was calculated from a standard curve prepared using crystalline sodium pyruvate. The kinetic parameters were computed from a Lineweaver–Burk transformation (V–1 versus S–1) of the Michaelis–Menten equation, where V (µmol min–1 mg–1) represented the formation of pyruvate and S (mM) was the concentration of each substrate. All values are reported as the means±SD of three independent experiments.
Real-time PCR analysis.
Each strain was grown in 40 ml BHI to an OD600 of about 1.0 (late exponential phase). Total RNA was then isolated from the harvested cells using FastPrep Blue tubes (Bio 101). Contaminating DNA was eliminated by digestion with RNase-free DNase (Takara Bio). RNA (10 ng) was reverse transcribed into single-stranded cDNA using PrimeScript Reverse Transcriptase (Takara Bio) according to the manufacturer's instructions. Real-time quantitative PCR amplification, detection and analysis were performed using the Thermal Cycler Dice RealTime System (Takara Bio) with Power SYBR Green PCR Master Mix (Applied Biosystems). Real-time PCR was carried out in 25 µl reaction mixtures (1x Power SYBR Green PCR Master Mix, 22.5 pmol of each forward and reverse primers and 2.5 µl template). The reaction conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. At the end of each run, a dissociation protocol (95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s) was performed to ensure that non-specific PCR products were absent. Each primer was designed using Primer Express software (version 3.0; Applied Biosystems). The amount of 16S rRNA was used as an internal standard to normalize for the amount of total RNA in each sample. The primers used to amplify the lcd gene were 5'-GGA CTT TGA AGT CAT GCC TGA AGT A-3' and 5'-CAC TGC TTG GAC TAG CTC ATC ACT-3', while those used for 16S rRNA were 5'-GGA CTT TGA ACT CAT GCC TGA AGT A-3' and 5'-CAC TGC TTG GAG TAG CTC ATC ACT-3'. To estimate the initial amounts of template in each sample, serial real-time PCR was performed using purified streptococcal genomic DNA. For each gene, a standard curve was plotted using the log of the initial quantity of template against the threshold cycle (i.e. the cycle at which the fluorescence rose above the background level). In this way, differences in primer efficiency could be accommodated. The data were obtained from three independent experiments.
High-performance liquid chromatography (HPLC).
Production of homocysteine or cysteine from cystathionine by βC–S lyase was determined on a reversed-phase column using HPLC. The reaction mixture contained the following reagents in a final volume of 100 µl: 50 mM potassium phosphate buffer (pH 7.6), 10 µM PLP, 2 mM L-cystathionine and 0.4 µg purified βC–S lyase from S. intermedius IMU201. After the mixture had been incubated for 6 h at 37 °C, the enzyme was removed using a Microcon YM-10 filter (10 kDa cutoff; Amicon). The ultrafiltration product was determined after derivatization with dansyl chloride as described by Tapuhi et al. (1981). An aliquot (20 µl) of the sample was injected onto an XTerra RP18 column (4.6x150 mm; Waters). A linear gradient (60–80 %) of methanol in distilled water containing 0.6 % (v/v) glacial acetic acid and 0.008 % (v/v) triethylamine was used at a flow rate of 1.0 ml min–1 at 40 °C. Excitation and emission wavelengths of 350 and 530 nm, respectively, were used.
Statistical analysis.
All data were analysed using Student's t-tests. A P value of <0.05 was considered significant.
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RESULTS AND DISCUSSION
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H2S production from L-cysteine in crude enzyme extracts prepared from S. intermedius
To examine H2S production in S. intermedius strains, crude enzyme extracts were obtained from one reference and 28 clinical strains of S. intermedius. H2S production from L-cysteine was generally lower than that of S. anginosus and S. constellatus (Fig. 2). In contrast, 26 of the 29 extracts showed a greater capacity for H2S production than in S. gordonii strains. Incubation of the three remaining crude extracts, which were derived from one reference and two clinical strains (strains ATCC 27335, IMU151 and IMU202), with L-cysteine did not produce H2S. These results were unexpected, because crude extracts from more than 25 streptococcal strains grown in BHI were able to produce H2S from L-cysteine. There are four possible explanations for the lack of detectable H2S production: (1) the existence of truncated protein encoded by a mutant lcd gene, (2) no (or extremely low) transcription of lcd, (3) no (or extremely low) enzymic activity of the purified Lcd for L-cysteine or (4) no (or extremely low) translation from mRNA of the lcd gene. These possibilities were evaluated in the following experiments.
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Fig. 2. H2S production in crude enzyme extracts from streptococcal strains. Each extract was incubated with L-cysteine (1 mM) for 10 min. The data are the means of three independent experiments. Bars indicate the mean level of H2S formation in each group. Activity data for S. anginosus, S. constellatus and S. gordonii (Yoshida et al., 2008) were compared to those for S. intermedius.
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Molecular analysis of the lcd gene encoding βC–S lyase in S. intermedius
To analyse the genetic basis of H2S production in S. intermedius, DNA fragments containing the lcd gene in seven S. intermedius strains, including four H2S-producing (IMU105, IMU122, IMU201 and DP102) and three non-producing (ATCC 27335, IMU151 and IMU202) strains were sequenced and analysed. The lcd homologues from the H2S-producing strains were 1164 bp long, which is identical to the length of the lcd genes in S. anginosus and S. constellatus. Database analysis revealed that the deduced amino acid sequences from these S. intermedius strains had high identity to those from 12 strains of S. anginosus and S. constellatus (90.5–94.1 %) (Yoshida et al., 2008). They also showed moderate sequence similarity with the amino acid sequences of four S. gordonii strains (75.0–75.5 %) (Yoshida et al., 2008). The Lcd protein from S. intermedius ATCC 27335 was shorter than those of the other strains, and primary sequence alignment revealed that 119 amino acids were missing from the C terminus of the S. intermedius ATCC 27335 Lcd protein. This mutation involved the deletion of a cytosine at position 809 in the lcd gene, which effectively changed a TCA (Ser) codon to a TAA terminator. A different mutation (the deletion of an adenine and a thymine at positions 180 and 181, which created a TGA terminator 13 bp further downstream) was, surpisingly, present in the lcd gene from both strain IMU151 and strain IMU202. Consequently, their deduced Lcd sequences lacked 47 amino acids at the N terminus, compared with the intact protein. Moreover, 1161 of the 1162 bp in these two truncated lcd genes were identical. These findings might suggest that these two strains had acquired the same truncated gene by lateral transfer. The presence of such mutations also suggests that selective pressure to maintain the complete gene may have been removed. If this were true, S. intermedius strains with disrupted lcd genes could be auxotrophs for methionine. To test this hypothesis, we attempted to grow these strains of S. intermedius in two different types of previously described chemically defined media (Socransky et al., 1985; Terleckyj et al., 1975), with or without methionine. However, even S. intermedius strains containing an active lcd gene failed to grow in these media. Thus, the effect of lcd disruption on auxotrophy in S. intermedius remains to be clarified.
Repair of the truncated lcd in S. intermedius ATCC 27335
We suspected that S. intermedius ATCC 27335 would have the capacity to produce H2S from L-cysteine following repair of the lcd pseudogene. Gene repair was accomplished in two steps (Fig. 3a). The repaired lcd contained cytosine at position 809, which was commonly identified in the other sequenced lcd genes of S. intermedius strains IMU105, IMU122, IMU201, IMU202, IMU151 and DP102. Integration of the overlap extension PCR products at the expected location in the chromosome was confirmed by amplification of the upstream and downstream boundaries of the insertion using primers specific for flanking sequences that were extraneous to those used for gene targeting (Fig. 3b). Repair of the lcd gene in strain KO101 was confirmed by sequencing of a PCR product amplified from the strain's genomic DNA.
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Fig. 3. Construction of a derivative of S. intermedius ATCC 27335 containing a repaired lcd gene. Each circled number indicates a primer used for verification. (a) Chromosomal gene arrangement in the parental and mutant strains. The size of each fragment is shown in base pairs. (b) Verification of the mutants by agarose gel electrophoresis. Each fragment was PCR amplified using the primers indicated. The positions of DNA size standards (kb) are shown.
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Incubation of crude extracts from strain KO101 with L-cysteine resulted in H2S production, indicating that the capacity of strain ATCC 27335 to produce H2S was restored by repair of the lcd gene (Fig. 4a). The rate of L-cysteine degradation in crude extracts prepared from strain KO101 was lower than that in extracts of S. anginosus and S. constellatus, but higher than that in extracts prepared from S. gordonii and similar to the rate in other S. intermedius strains (Fig. 2). Furthermore, the restoration was confirmed by in situ staining, in which H2S is allowed to react with bismuth to produce an insoluble product that forms brown bands on non-denaturing gels. A single band associated with the production of H2S was detected in crude extracts prepared from strain KO101, whereas strains ATCC 27335 and KO100 showed no detectable bands (Fig. 4b). These findings demonstrate that no other enzymes are responsible for the production of H2S and that the frameshift in the lcd gene abolished H2S production in ATCC 27335. It is possible that repair of the lcd genes in strains IMU151 and IMU202 might also restore H2S production.
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Fig. 4. Restoration of H2S production by repair of the lcd gene in S. intermedius ATCC 27335. (a) H2S production from 1 mM L-cysteine with crude extracts. The values represent the means±SD of three independent experiments. (b) Electrophoresis of crude extracts of the parental and mutant strains of S. intermedius. Left panel: the samples (45 µg) were subjected to SDS-PAGE, and the gel was subsequently stained with Coomassie brilliant blue. Right panel: the samples (45 µg) were subjected to non-denaturing PAGE, and enzymic activity was visualized. Lanes: 1, S. intermedius ATCC 27335 cell lysate; 2, S. intermedius KO100 cell lysate; 3, S. intermedius KO101 cell lysate. The positions of molecular mass markers (kDa) are shown.
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Quantification of lcd transcription in S. intermedius
The fact that we were able to restore H2S production by lcd gene repair suggests that lcd transcription was not interrupted in strain ATCC 27335. Real-time PCR analysis was used to evaluate the relative abundance of lcd-bearing transcripts among strains ATCC 27335, IMU151 and IMU202 (with an inactive lcd gene), KO101 (with a repaired lcd gene) and IMU201 (with an active lcd gene). To estimate the initial quantity of template in each sample, tenfold serial dilutions of genomic DNA were prepared to generate a standard curve using primers to amplify lcd (Fig. 5a) and 16S rRNA (data not shown). The purity of the amplified products was assessed by dissociation curve analysis (Fig. 5b) and electrophoresis on 1.8 % (w/v) agarose gels (data not shown). As shown in Fig. 5(c), these strains did not significantly differ in lcd transcript abundance, suggesting that the mutation in lcd had no effect on gene expression in strain ATCC 27335.
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Fig. 5. Real-time quantitative PCR analysis. (a) Standard curve. The reaction mixture contained the primers used for lcd amplification and genomic DNA extracted from S. intermedius ATCC 27335. The standard curve is a plot of the initial template quantity (x-axis) versus the threshold cycle (R2=0.999). (b) Dissociation curve analysis of the real-time PCR products used to prepare the standard curve shown in (a). The plot was based on the first derivative of the fluorescence reading. The specific PCR products melted at 77±1 °C. (c) Comparison of lcd expression among S. intermedius IMU201, ATCC 27335, KO101, IMU151 and IMU202. The amount of lcd cDNA was determined and normalized against the amount of 16S rRNA cDNA following reverse transcription of total RNA. The expression of lcd in strains ATCC 27335, KO101, IMU151 and IMU202 is indicated relative to that in strain IMU201. The data are the means±SD of three experiments; the cDNA used in each experiment was generated from independently isolated RNA.
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Purification and enzymic characterization of recombinant Lcd
To characterize the enzymic activity of the lcd products, recombinant Lcds from S. intermedius strains ATCC 27335, KO101 and IMU201 were purified. Each purified protein was obtained by cleavage of the GST-fusion protein bound to glutathione-Sepharose 4B. SDS-PAGE was used to assess the purity of each recombinant Lcd (Fig. 6). The observed size of the proteins was consistent with their predicted molecular masses (31 kDa for ATCC 27335 and 44 kDa for KO101 and IMU201). The reason why the sample from ATCC 27335 contained unexpected faint bands is unknown. The same purification method failed to isolate the other truncated lcd product of S. intermedius IMU202, whose amino acid sequence was identical to that of S. intermedius IMU151. Since the GST-Lcd fusion protein of strain IMU202 was largely produced in E. coli cells, PreScission protease might not recognize the core amino acid sequence for cleavage between GST and Lcd. Indeed, substrate recognition and cleavage are likely to be dependent not only upon primary structural signals, but also upon the secondary and tertiary structures of the fusion protein (manufacture's protocol). Alternatively, the GST-Lcd fusion protein might be insoluble due to formation of inclusion body in host cells.
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Fig. 6. SDS-PAGE analysis of recombinant purified lcd products from the S. intermedius strains. The samples were subjected to SDS-PAGE and the gel was stained with Coomassie brilliant blue. Lanes: 1, recombinant Lcd from S. intermedius ATCC 27335; 2, recombinant Lcd from S. intermedius KO101; 3, recombinant Lcd from S. intermedius IMU201. The positions of molecular mass markers (kDa) are shown. Arrows indicate the 44 and 31 kDa bands of the predicted sizes.
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Some βC–S lyases have been reported not only to cleave cystathionine to yield ammonia, pyruvate and homocysteine via ,β-elimination, but also to degrade cystathionine to ammonia, -ketobutyrate and cysteine via ,-elimination (Alting et al., 1995; Dobric et al., 2000) To determine whether Lcd of S. intermedius catalyses ,-elimination, HPLC analysis was carried out (Fig. 7). Each amino acid was detected as a dansylated product. Incubation of Lcd from S. intermedius IMU201 with cystathionine resulted in production of homocysteine, while no production of cysteine was detected. These results demonstrated that Lcd from S. intermedius catalyses only ,β-elimination. Dansylhomocysteine was detected at 12.97 and 13.56 min in this condition. This may be due to the disulfide formation by oxidation of SH-compounds (Schulze & Neuhoff, 1976).
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Fig. 7. Reversed-phase HPLC profiles of dansylated reaction products. (a) A mixture of pure standard chemicals. (b) Reaction products obtained with no enzyme (negative control). (c) Reaction products obtained with purified Lcd from S. intermedius IMU201. Peaks were identified by retention time.
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The breakdown of L-cysteine and L-cystathionine was monitored by assaying the production of pyruvate, which is a by-product of the reactions that degrade these substrates. The truncated Lcd of S. intermedius ATCC 27335 was unable to degrade L-cysteine or L-cystathionine (data not shown), suggesting that both the trans-sulfuration and direct sulfhydrylation pathways may be interrupted in this strain (Fig. 1). In contrast, the protein encoded by the repaired lcd in strain KO101 had a slightly higher capacity to degrade L-cysteine and L-cystathionine than the Lcd from strain IMU201, which contained an active lcd. The kinetic properties of the Lcds from S. intermedius KO101 and IMU201, which were calculated from Lineweaver–Burk plots, are summarized in Table 2. The Km values of the Lcds from S. intermedius KO101 and IMU201 for L-cysteine (0.26 and 0.43 mM, respectively) were lower than those for the type strains of S. anginosus and S. constellatus (0.99 and 0.67 mM, respectively), indicating that the Lcd from S. intermedius has a high affinity for L-cysteine. In terms of the Km value for L-cystathionine, the S. intermedius strains were comparable to S. anginosus and S. constellatus (0.41 and 0.71 mM, respectively). Thus, both the trans-sulfuration and direct sulfhydrylation pathways of methionine biosynthesis may be at work in S. intermedius. Kredich (1996) described that the direct sulfhydrylation pathway may be physically irrelevant due to the high concentration of sulfide required. In this regard, it is noteworthy that the βC–S lyase in S. intermedius, as well as those in S. anginosus and S. constellatus, was shown to have an extremely high capacity for H2S production from L-cysteine compared to other oral streptococci, including S. gordonii, S. oralis, S. mutans, S. sobrinus and S. salivarius (Yoshida et al., 2003a, 2008).
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Table 2. Kinetic properties of the lcd products from S. intermedius strains
Values are given as the means±SD of three determinations.
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Concluding remarks
H2S is widely known as a toxic gas with the smell of rotten eggs. It was recently discovered, however, that the gas is also associated with endotoxin-induced inflammation (Li et al., 2005) and apoptosis (Yang et al., 2004). Therefore, H2S production in anginosus group streptococci may be associated with abscess formation, based on previous research showing that these bacteria are frequently encountered in suppurative infections in a variety of clinical sites, such as liver and brain abscesses (Gossling, 1988; Jacobs et al., 1995; Molina et al., 1991; Van der Auwera, 1985; Whiley et al., 1992). In this regard, it is notable that the three S. intermedius strains containing a disrupted lcd gene (ATCC 27335, IMU202 and IMU151) were not isolated from abscesses. The type strain ATCC 27335 isolated some decades ago has been maintained since then in a laboratory environment. Indeed, there was no significant difference in growth among S. intermedius strains ATCC 27335, KO100 and KO101, when incubated in BHI, which appears to fulfil the nutritional requirements of the bacteria (data not shown). In contrast, two clinical strains with the disrupted lcd may occupy an ecological niche where methionine biosynthesis and/or H2S production is unnecessary. However, critical evidence of a relationship between elevated H2S production and abscess formation is lacking. Additional studies on abscess formation by H2S-producing or non-producing strains are therefore necessary.
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ACKNOWLEDGEMENTS
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We are grateful to Drs Kazushi Kunimatsu (Department of Periodontology, Iwate Medical University School of Dentistry) and Hirohisa Kato (Department of Dental Pharmacology, Iwate Medical University School of Dentistry) for their contributions during preparation of this manuscript. We also thank members of the Central Clinical Laboratory, Iwate Medical University Hospital, for collecting the clinical strains. This study was supported in part by a grant from the Keiryokai Research Foundation, no. 2006-96 (Y. Y.), by a Research Grant for Promoting Technological Seeds (Y. Y.) and by Grants-in-Aids for Scientific Research no. 18592004 (H. N.) and no. 20592463 (Y. Y.) and for High-Tech Research Project (2005–2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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INTRODUCTION
METHODS
