INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Lactate dehydrogenase (LDH)-deficient mutants of mutans streptococci have been studied for their potential use in the replacement therapy of dental caries (10). Without LDH, fermentation of carbohydrates by this microorganism occurs via alternate pathways for pyruvate metabolism that yield significant amounts of neutral end products and smaller amounts of total acids (6). As a result, LDH-deficient mutants are less cariogenic. In addition to being less cariogenic, an effector strain for replacement therapy of dental caries must demonstrate superior colonization properties to displace indigenous mutans streptococcal strains which may be present in the host while preventing subsequent colonization by wild-type strains whenever the host is exposed to them.

Numerous studies (13, 14, 22, 25, 26) have documented the difficulty of introducing mutans streptococci into the mouths of humans, particularly if they already harbor an indigenous strain of this organism. We previously reported the isolation from a clinical sample of a strain of Streptococcus mutans called JH1000, which has unusually good colonization properties (9). This strain was found to produce a potent, broad-spectrum bacteriocin-like inhibitory substance (BLIS). In a deferred antagonism assay, the JH1000 BLIS was found to inhibit the growth of representative strains of S. salivarius, S. sanguis, S. oralis, S. mitis, S. pyogenes, Staphylococcus aureus, Lactobacillus salivarius, L. casei, Actinomyces israelii, A. naeslundii, and A. viscosus. In addition, virtually all 124 mutans streptococcal strains tested, including both fresh isolates and laboratory strains, were sensitive. Preliminary studies performed on crude BLIS preparations indicated that the active component was both small (molecular weight, <1,000) and proteinaceous.

Analysis of isogenic mutants demonstrated a good correlation between BLIS production and colonization potential in both a rodent model (9) and human subjects (8, 11). A mutant called JH1005, which produces ca. threefold-elevated levels of BLIS activity, could be recovered from saliva samples 3 years after brushing and flossing it onto the teeth of human subjects during a single, 3-min infection regimen (7, 8). During that period, the proportion of indigenous S. mutans cells recoverable from the same plaque samples decreased to undetectable levels. Based on these and other results, it was proposed that an LDH-deficient mutant of JH1005 would possess the necessary combination of properties, including low virulence and superior colonization potential, to serve as an effector strain in the replacement therapy of dental caries.

Previous, unreported attempts involving standard biochemical methods to isolate and characterize the JH1005 BLIS activity were unsuccessful, probably because its production is less reliable when cells are grown in liquid medium than when they are grown in solid medium. In this paper, we describe genetic methods which enabled us to identify the BLIS activity and to devise methods for its large-scale purification. It was found to be a lantibiotic with significant homology to epidermin produced by Staphylococcus epidermidis and to the recently reported lantibiotic from S. mutans Ny266 (18).

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Organisms and culture conditions. S. mutans JH1005 and JH1140 are, respectively, ethyl methanesulfonate-induced and spontaneous mutants of JH1000 that show three- to fourfold-increased BLIS activity (9). Strain DM25 is a Tn917-induced BLIS-negative mutant of JH1005 (5). S. rattus BHT-2 (resistant to 1 mg of streptomycin sulfate ml¹ [9]) was used as a target strain in BLIS purification assays. Other bacterial strains are listed in Table 1. For routine cultivation, brain heart infusion (BHI) broth and agar (1.5%) (Difco Laboratories, Detroit, Mich.) were used. For determination of auxotrophic properties, the minimal medium of Carlsson (2) was used.

Tn917 mutagenesis and recovery of interrupted DNA. Plasmid pTV1-OK is a repA(Ts) derivative of the Lactococcus lactis cryptic plasmid pWV01 for temperature-dependent replication in both S. mutans and Escherichia coli (5). It possesses a kanamycin resistance (Kan^r) gene, which functions in both E. coli and S. mutans, and transposon Tn917, which confers erythromycin resistance (Em^r) in streptococci and in E. coli MC1061. Transposon mutagenesis of strain JH1005 harboring pTV1-OK was performed by the method of Gutierrez et al. (5). After a temperature shift to eliminate the plasmid, Em^r clones were selected on BHI agar containing 15 µg of the antibiotic ml¹. These were stab inoculated (25 stabs/plate) into the same medium without antibiotic. After incubation overnight in candle jars at 37°C, the plates were overlaid with 3 ml of top agar containing ca. 10⁵ CFU of BHT-2 ml¹. Stabbed clones which failed to produce a zone of growth inhibition in the BHT-2 lawn in this deferred-antagonism assay were recovered and purified by streaking on medium containing erythromycin.

1

1

Other genetic methods. Southern analysis was performed with pTV1-OK as a probe and EcoRI-digested chromosomal DNA. The enhanced chemiluminescence gene detection system was used as specified by the manufacturer (Amersham International PLC, Little Chalfont, England). Transformation of S. mutans was carried out by the method of Perry and Kuramitsu (21). Other DNA manipulations were as described by Maniatis et al. (16).

Purification and characterization of the JH1140 BLIS. Large-scale preparations of BLIS were performed with Todd-Hewitt broth (Difco) containing 0.5% SeaKem LE agarose (FMC BioProducts, Rockland, Maine). Batches of this medium (4 liters) in petri dishes were stab inoculated with a 10-prong replicator that was first stabbed into a BHI plate with a confluent lawn of JH1140. The plates were incubated in candle jars for 72 h at 37°C. The agar was scraped from the plates, aliquoted into centrifuge bottles, and frozen overnight at 20°C. The bottles were centrifuged at 4,000 × g for 60 min and then at 8,000 × g for 30 min at room temperature. The resulting supernatant was passed through Whatman no. 1 filter paper to remove agar fines. Chloroform (33.3 ml liter of culture supernatant¹) was added, and the mixture was vigorously agitated for 2 h with a magnetic stirrer. After overnight standing at room temperature, the aqueous phase was removed by aspiration. The remaining chloroform layer, containing a white precipitate, was centrifuged at room temperature for 8 min at 2,500 × g. The chloroform was decanted, and the precipitate was isolated, washed twice by centrifugation with 10 ml of chloroform, and dried under a stream of nitrogen in a 45°C water bath. A 1-ml volume of 50% ethanol was added to the resulting residue and vortexed vigorously for 15 s. Undissolved material was removed by centrifugation at 14,000 × g for 1 min at room temperature. The supernatant, which we called fraction A, was stored at 4°C.

Assay of mutacin 1140 activity. Two methods were used to determine the presence of mutacin 1140 activity. In the quantitative method, samples (25 µl) of fractions to be tested for mutacin 1140 activity were serially twofold diluted in distilled and deionized H₂O in 96-well microtiter plates. Top agar (BHI broth containing 0.75% agar) was melted and then cooled to 42°C in a water bath. An overnight culture of BHT-2 grown in BHI broth was added to the top agar to give a final concentration of ca. 10⁵ CFU ml¹. A stock solution of streptomycin sulfate was also added to give a final concentration of 1 mg ml¹. Aliquots (200 µl) of the top agar were then added to the samples in the microtiter wells. After gelling, the plates were sealed with Mylar and incubated inverted at 37°C for 24 h. Growth of BHT-2 was determined for each well under ×10 magnification, and the activity titer for each sample tested was calculated as the reciprocal of the highest dilution which inhibited growth.

1

Nucleotide sequence accession number. The GenBank/EMBL accession number for the nucleotide and deduced amino acid sequences of the genes described here is AF051560.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Genetic studies. A culture of S. mutans JH1005, transformed with the Tn917-bearing, temperature-sensitive plasmid pTV1-OK, was shifted to 42°C to eliminate the plasmid. The frequency of transposition (Em^r) was found to be ca. 10⁴ cell¹, and the frequency of replicon fusion (integration of the entire plasmid into the chromosome; Em^r Kan^r) was ca. 10⁶ cell¹. A total of 750 independent Tn917 transposon mutants of JH1005 were individually analyzed for BLIS production by determining their ability to inhibit the growth of the S. rattus target strain BHT-2 in the deferred-antagonism assay. One BLIS-negative mutant, DM25, was isolated in this fashion.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   lan gene cluster. (A) Diagram of the orientation and relationship of the four open reading frames and the site of Tn917 insertion (). (B) Nucleic acid sequence of the JH1005 DNA insert in pDM25, showing the deduced amino acid sequences encoded by the open reading frames, putative stem-loop structures, putative transcriptional and translational regulatory sequences, the site of cleavage of the prelantibiotic (), the partial gene duplication in lanA and orfY, and the site of Tn917 insertion ().

Purification and characterization of the JH1140 BLIS. Although JH1005 and JH1140 make approximately threefold-elevated amounts of BLIS compared to their parent, JH1000, broth culture liquors of these strains did not contain reliably high levels of activity. Consequently, we attempted to purify the putative lantibiotic indicated by the previous studies by growing JH1140 on solid medium consisting of Todd-Hewitt broth containing 0.75% agar. Plates that were stab inoculated with JH1140 and incubated for 3 days showed large (>25-mm) zones of inhibition when overlaid with BHT-2. When the fluid phase of the agar was extracted by freeze-centrifugation and treated with chloroform, no activity was associated with the resulting precipitate, as would be expected for a lantibiotic (19). JH1140 produced comparable zones of growth inhibition on medium in which agarose (0.5%) was substituted for agar. Chloroform treatment of the fluid extracted from agarose-containing medium produced a precipitate which, when dissolved in 50% ethanol (fraction A), did contain large amounts of an activity that inhibited the growth of BHT-2 and other strains sensitive to the JH1140 BLIS (Table 1). This result suggested that agar contained a contaminant which bound the active BLIS component during the purification procedure and which was absent in agarose.

View larger version (100K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE analysis of fraction A from the parent and mutant. Chloroform treatment of JH1140 and DM25 culture liquors yielded precipitates, which were dissolved in 50% ethanol to yield fraction A. Samples were electrophoresed on a Tris-Tricine-10 to 20% polyacrylamide gradient gel and stained with Coomassie brilliant blue. Std, protein molecular weight standard.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Proposed structure of mutacin 1140 and comparison to B-Ny266 and epidermin. Abbreviations: dha, 2,3-didehydroalanine; dhb, 2,3-didehydrobutyrine; abu, 2-aminobutyric acid.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Because strain JH1005 is poorly transformable, it was not possible to create a library by conventional insertional inactivation methods to screen for BLIS mutants. We have previously described the construction and successful testing of the thermosensitive vector, pTV1-OK harboring Tn917, as a tool for random mutagenesis in poorly transformable strains of S. mutans (5) and other streptococci. Previously, we reported the isolation of a Tn917-induced BLIS-negative mutant which proved to be defective in formyl-tetrahydrofolate synthetase (3). In the present study, we isolated another BLIS-negative mutant, DM25, that allowed us to identify the BLIS activity as a lantibiotic called mutacin 1140.

Marker rescue of the DM25 Tn917 insertion yielded a pUC19-based plasmid containing a total of 2,086 bp of DNA 5' and 3' to the transposon. Four potential open reading frames were identified, two of which (orfX and orfY) had no homology to database sequences. The remaining two open reading frames had extensive homology to the structural gene and a gene encoding a modifying enzyme for the S. epidermidis lantibiotic epidermin.

The BLIS-negative phenotype of DM25 cannot be clearly explained by the location of Tn917 in orfY, which has no apparent ribosome binding site. If orfY is expressed, it may be from a nonstandard start site upstream or downstream of the indicated ATG, or there may be a nonstandard ribosome binding site. Expression of orfY may occur in the absence of a ribosome binding site by ribosomal hopping, as described by Farabaugh (4), from lanA, the end of which is only 27 bp removed from the start of orfY. Polar effects of Tn917 on lanB could be invoked, even though this gene appears to have its own promoter based on sequence analysis. It remains to be determined whether lanB is part of the lanA transcript or is transcribed separately.

A large number of BLIS activities from a variety of species have been difficult to purify and characterize owing largely to their poor production in liquid medium. We have preliminary data (27) which indicate that, in the case of mutacin 1140, this is due in part to inhibition of transcription of the structural gene by the mature lantibiotic. We hypothesize that diffusion of the lantibiotic away from stab-inoculated cells growing on solid medium permits a greatly extended period of synthesis before a threshold inhibitory concentration is reached.

However, recovery of mutacin 1140 from solid medium in practical amounts was not possible until we replaced standard cultivation agar with agarose. Comparable inhibition zone sizes were observed when an S. rattus target strain was overlaid on either agar or agarose plates stabbed with JH1140. Hence, agar did not affect the synthesis or stability of mutacin 1140 but must have bound it in a fashion that prevented its extraction into chloroform without altering its biological activity. We did not examine the possibility of eluting mutacin 1140 from agar, which might have served as a practical purification step. Rather, we found that substitution of agarose for agar permitted the recovery of mutacin 1140 from freeze-centrifugation extracts where it constituted one of only two major protein bands as seen on polyacrylamide gels. Perhaps as many as 20 additional proteins were also present in small or trace amounts.

The amino acid sequence of purified mutacin 1140 as determined by Edman analysis and MS corresponded to that deduced from the lanA sequence. The observed molecular weight of 2,263 is considerably larger than that predicted in previous studies (9), in which mutacin 1140 was found to pass through dialysis membranes with a molecular weight cutoff of 1,000. Also, the migration of mutacin 1140 in SDS-PAGE analysis was slower than expected based on its known molecular weight (Fig. 2). We believe that both of these apparent discrepancies are because mutacin 1140, like other lantibiotics (23), possesses a somewhat rigid, rod-like structure that would enhance its ability to move through dialysis membrane pores and retard its electrophoretic mobility.

The deduced primary amino acid sequence of the lanA leader sequence showed limited homology to the leader sequences of other reported lantibiotics (Fig. 4). However, 16 of the 22 amino acids that form the mature mutacin 1140 are identical to the epidermin sequence and 2 others have conserved homology (Fig. 3). Based on the proposed structure of the S. mutans lantibiotic B-Ny266 (18), mutacin 1140 is probably more closely related to this molecule. Since Edman sequencing of ethanethiol-treated lantibiotics does not permit assignment of a position for Cys and Ser residues involved in the formation of lanthionine, and in the absence of genetic data, the precise primary amino acid sequence and secondary structure of B-Ny266 are unknown. However, it is possible that B-Ny266 differs from mutacin 1140 in as few as two positions, i.e., Phe versus Leu at residue 6 and Lys versus Arg at position 13. Both of these differences can occur as a result of single-base changes in the lanA nucleotide sequence.

View larger version (9K): [in this window] [in a new window]   FIG. 4.   Multiple amino acid sequence alignment of the leader sequences of mutacin 1140 and other lantibiotics. Sequence homology alignment was performed with the CLUSTAL-W program. Mutacin 1140 residues that are identical to other aligned lantibiotic residues are indicated by boxes.

The proposed secondary structure of mutacin 1140 differs significantly in the N-terminal half of the molecule from that proposed for B-Ny266 and from the established structure for epidermin (Fig. 3). Our MS data strongly support the smaller and more widely separated ring structures as depicted. Differences in the ring structures of the C-terminal half of the molecules may also exist, but our MS data were not able to provide a definitive structure for this portion of mutacin 1140. Additional data from nuclear magnetic resonance spectroscopy analysis are being gathered to answer this question. The variation between mutacin 1140 and B-Ny266 or epidermin in the positions of their thioether bridges and didehydro amino acids probably reflects differences in the specificities of the modifying enzymes, although it is also possible that the several differences in their amino acid sequences are responsible. It would be of interest to put the lanA structural gene into an appropriate Ny266 or S. epidermidis background to clarify this point.

A novel feature of the proposed mutacin 1140 structure is that the Cys-derived "half" of the methyllanthionine (residues 11 to 14) is located on the N-terminal side of the diamino acid while the Thr-derived half is located toward the C terminus. This has not been observed previously among the type A lantibiotics (23). Also, the deduced lanA polypeptide does not show a consensus sequence surrounding the point of cleavage between the leader peptide and the propeptide (Fig. 3 and 4). Unlike other reported lantibiotics, which contain hydrophobic amino acids in positions 4 and +2 and Pro in position 2, mutacin 1140 contains Pro, Lys, and Thr, respectively. This finding differs from results for previously reported lantibiotics and suggests the possibility that processing of mutacin 1140 differs from that of other known lantibiotics.

We have discovered by Edman degradation following ethanethiol derivatization that the other major protein present in chloroform precipitates of JH1140 culture liquors has a sequence that is identical to the faster-migrating band, which we know from the above studies to be the mature lantibiotic mutacin 1140. No additional residues were found at either the amino- or the carboxy-terminal end of the molecule. This result has been confirmed by MALDI-TOF MS of the blotted band, which showed a peak with a molecular weight of 2,263, along with several other minor contaminant peaks. This observation is difficult to explain: the slower band may be a partially or wholly unmodified or differently modified molecule, reflecting the Tn917 insertion which knocks out expression of one or more enzymes involved in processing the prepropeptide. While this would be expected to result in differences in apparent molecular weight by SDS-PAGE analysis, we would also expect differences in the Edman sequencing, which were not observed. We anticipate that further MS analyses will help to resolve this question.

Mutant analysis of JH1000 provided the first strong evidence for the role of a BLIS activity in promoting the colonization of a susceptible host by a producer strain (9). The identification here that the JH1000 BLIS is a lantibiotic accords well with our proposed scheme to use a JH1000 derivative as an effector strain for replacement therapy of dental caries. Nisin, the prototype lantibiotic, has low toxicity (12) and has been used widely for food preservation. Other lantibiotics are also being developed for use in clinical treatment of infections and other applications.