Diacylglycerol Kinase Is Involved in Regulation of Expression of the Lantibiotic Mutacin II of Streptococcus mutans

Abstract

Genetic characterization of a Tn916 transposon mutant, Streptococcus mutans T8-1, defective in mutacin II production, revealed that the transposon was inserted into the 3′ region of a diacylglycerol kinase (dgk) gene. The insertion occurred in the same region as described for another S. mutans mutant, GS5Tn1, which was altered in its ability to respond to environmental stress (Y. Yamashita, T. Takehara, and H. K. Kuramitsu, J. Bacteriol. 175:6220–6228, 1993). Quantitative primer extension from the mutacin structural gene mutA showed a decreased level (about eightfold) of mutA transcription for mutant T8-1. Mutacin production was restored by transforming mutant T8-1 with integration vector pVA891 containing an intact dgk gene. These data indicated that the full-length dgk gene product along with the mutacin biosynthetic operon are required for the production of the mutacin II lantibiotic.

Streptococcus mutans has been implicated as a major etiologic agent in human dental caries (17). One major factor thought to be involved with the ability of S. mutans to colonize tooth surfaces is the production of mutacins (11), or bacteriocin-like inhibitory substances (15, 24), active against other gram-positive bacteria found in plaque biofilms (12, 24, 32). One subset of mutacins, designated mutacin II, is elaborated by group II S. mutans strains, including UA96 and T8, and has been isolated and partially characterized (6, 20, 21). Mutacin II is a 3,245-Da peptide exhibiting stability and antimicrobial activity over a wide range of pHs and temperatures. Comprising two lanthionines, one methyllanthionine, and a didehydroamino acid, mutacin II belongs to a group of bacteriocins called lantibiotics, which are ribosomally synthesized and undergo several posttranslational modifications (25, 26). Mutacin II is bactericidal to gram-positive bacteria by inhibiting their energy metabolism, an activity not reported for other known lantibiotics (6).

Recently, part of the mutacin II biosynthetic operon, mutA and mutM, which encodes the prepromutacin and the modification enzyme, has been cloned and sequenced (34). In addition to the biosynthetic operon, additional loci are also likely required for mutacin production, as indicated by the identification of at least five different mutants generated by transposonal mutagenesis (5).

To characterize these loci, we used a single-specific-primer PCR (SSP-PCR)-based technique (22) for isolating DNA adjacent to the Tn916 insertion. Characterization of one of these mutants has led to the identification of one of the essential loci, the diacylglycerol kinase (DGK) gene (dgk) locus. Our data suggest that DGK plays an important role not only in adaptation to environmental stress (35) but also in mutacin II production in S. mutans T8.

Bacterial strains, plasmids, and culture conditions.

Bacterial strains and plasmids used in this study are listed in Table 1. The Escherichia coli strains used for subcloning and plasmid isolation were grown in Luria-Bertani medium in the presence of the appropriate antibiotics. S. mutans strains and Streptococcus sobrinus OMZ176 were stored frozen at −70°C until needed and grown in Todd-Hewitt broth as described before (5, 20). TSBY/CDM medium (20) was used to grow S. mutans for the isolation of RNA for primer extension experiments.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Relevant characteristicsa	Reference or source
E. coli strain
INVαF′	F′ endA1 recA1 hsdR17(rK− mK+) supE44 thi-1 gyrA96 relA1 φ80lacZΔM15 Δ(lacZYA-argF)U169 λ−	Invitrogen, San Diego, Calif.
S. mutans strains
UA55	Tcr, Tn916 inserted in DGK, Mut−	2a
T8	Wild type, Mut+	23
T8-1	Tcr, Tn916 inserted in DGK, Mut−	This study
T8-1T	Tcr Emr, T8-1 transformed with pCBD5, Mut+	This study
S. sobrinus strain
OMZ176	Mutacin-sensitive indicator	5
Plasmids
pUC19	AprLacZa	36
pCRII	Apr Kmr, cloning vector	Invitrogen
pNoTA/T7	Apr, cloning vector	5 Prime → 3 Prime, Inc., Boulder, Colo.
pVA891	Emr Cmr, shuttle plasmid	18
pCBD1	Apr Kmr, pCRII-containing 2.5-kb TnLO-2 SSP-PCR fragment containing sgp	This study
pCBD2	Apr Kmr, pCRII with 0.8-kb Tn-R-O SSP-PCR fragment containing truncated dgk and part orf3	This study
pCBD3	Apr Kmr, pCRII with 0.9-kb SSP-PCR fragment containing the promoter region and 5′ end of orf3	This study
pCBD4	Apr, pNoTA/T7-containing 0.6-kb PCR fragment containing complete dgk coding region	This study
pCBD5	Emr Cmr, pVA891-containing 0.6-kb PCR fragment containing complete dgk coding region	This study
pCBMA	Apr, pNoTA/T7-containing 2.2-kb PCR fragment containing complete mutA coding region	5a

^aAbbreviations: Ap, ampicillin; Em, erythromycin; Km, kanamycin; Tc, tetracycline; Mut⁻, mutacin negative; Mut⁺, mutacin positive. 

Characterization of the Tn916 insertion region.

The broad-host-range conjugative transposon Tn916, originally identified on the chromosome of Enterococcus faecalis (9), has been used as a mutagen in a wide variety of bacteria including streptococci (3, 35), clostridia (2), and neisseriae (14). Several loci implicated in the production of the lantibiotic mutacin II from S. mutans UA96 were identified by this strategy (5). Recently, another mutacin II-defective mutant, S. mutans UA55, was generated. S. mutans UA55 is a transposon-containing non-mutacin-producing mutant of the parental strain UA96. Strain T8-1 was constructed by backtransforming the chromosomal DNA of the original strain, UA55, into the host strain, T8. Southern blotting ensured the presence of a single copy of the Tn916 transposon in the chromosomal DNA. The strategy for cloning of the Tn916 insertion region is illustrated in Fig. 1. Chromosomal DNA from strain T8-1 was isolated and digested with the restriction endonuclease HindIII and then ligated into HindIII-digested pUC19. The ligation mixture served as the template for SSP-PCR (22, 28, 29) with transposon-specific primers designed toward the left (TnLO-2 [5′-GTGAAGTATCTTCCTAC-3′]) or right (Tn-R-O [5′-TGAGTGGTTTTGACC-3′]) end of Tn916 (7). A 2.5-kb fragment was generated from the TnLO-2 and F-20 primer set. The region upstream of the right end of Tn916 was amplified by using the same technique with the Tn-R-O and F-20 primer set, except that the chromosomal DNA was digested with XbaI and ligated into XbaI-cut pUC19. Sequence analysis (see Fig. 2) indicated that a GTP-binding protein was located downstream of the left end of the inserted Tn916, while the upstream region of the right end of Tn916 encoded a DGK. Two additional primers were designed according to the newly available DNA sequence, and the original dgk locus was PCR amplified from the wild-type strain, T8, and sequenced. Comparison of the sequence adjacent to the Tn916 insertion region of T8-1 with the wild-type sequence in T8 showed that Tn916 was inserted within the codon of the eighth amino acid from the C terminus of DGK. Interestingly, the Tn916 insertion site in T8-1 was in the same region as observed in S. mutans GS5Tn1, with Tn916 in the same orientation (35). Yamashita and coworkers (35) showed that this insertion in S. mutans GS-5 resulted in lost adaptability to environmental changes. The sequences around the two insertion sites are slightly different; however, the general feature of the Tn916 insertion site characterized by high TA content is preserved. In fact, the deduced amino acid sequence from S. mutans T8 in this insertion region is the same as that from S. mutans GS-5 except for one amino acid difference in DGK at position 10 from the C terminus (V in T8 and I in GS-5).

FIG. 1.

Diagram of the dgk locus from S. mutans T8 and the cloning strategy. The site of the Tn916 insertion in S. mutans T8-1 is indicated by the black triangle. Only the relevant parts of clones pCBD1 and pCBD3 are shown. The promoter (P) and direction of transcription of the respective genes are indicated by arrows.

FIG. 2.

Nucleotide sequence and deduced amino acid sequence of the dgk locus. A putative promoter sequence (−35 and −10 regions) and ribosome binding site (RBS) are shown. Inverted repeat sequences are indicated by the dashed arrows. The transcription of orf3 starts 24 nucleotides upstream of the translation initiation codon. The site of the Tn916 insertion in the 3′ end of the S. mutans T8-1 dgk gene is indicated by the triangle.

Identification of the putative operon in which Tn916 was inserted.

Using the SSP-PCR chromosomal walking technique, the 5′ portion of orf3 and its upstream region was cloned (Fig. 1). In addition to the DGK and G-protein, this operon encodes a third hypothetical protein, ORF3, of unknown function. This is consistent with an earlier study (35). A hydrophobicity plot (data not shown) of ORF3 did not reveal any obvious signal sequence or hydrophobic regions, suggesting that it may be a cytoplasmic protein. Analysis of the putative protein encoded by orf3 did not uncover any apparent functional motifs. A BLAST search indicated that proteins similar to ORF3 also exist in Bacillus subtilis (YqfG), E. coli (U82598), Haemophilus influenzae (HI0004), Mycobacterium leprae (B1937_F1_21), Mycoplasma genitalium (MG388, U02265), Mycoplasma pneumoniae (AE000027), Serpulina hyodysenteriae (X73141) and Synechocystis sp. (D64001). The orf3 and dgk genes appear to be organized in similar fashions (adjacent to each other) in B. subtilis and Synechocystis. Sequence alignment indicated several conserved regions, including three histidines at the C terminus (data not shown). Considering the existence of similar proteins in other species, even in M. genitalium, which is thought to contain the smallest genome for a self-replicating organism, and our unsuccessful efforts to inactivate ORF3, orf3 may function as an essential housekeeping gene.

To locate the promoter(s) in this operon, total RNA from T8 wild-type cells in the early stationary growth phase was isolated with the hot phenol extraction method (19) and used for primer extension mapping (16) with a primer (5′-AGTAACCGCCATTTCTTTGTCTTC-3′) which is complementary to codons 34 to 41 of ORF3. The results (not shown) indicated that transcription of this operon was initiated at the A residue 24 bp upstream of the translation initiation codon for ORF3. By searching the DNA sequence upstream of the transcription start site, a putative −10 region, which has the sequence TATAAT and is located 6 bp from the transcription start site, was found (Fig. 2). Separated from the −10 region by 17 bp is a putative −35 region with the sequence TTAGAA. There are also minor read-through activities from the upstream promoter(s).

The effect of the truncated DGK on transcription of the mutacin structural gene mutA.

To address whether transcription of the mutacin structural gene mutA was affected by the Tn916 insert, resulting in the truncation of DGK, a quantitative primer extension analysis of mutA transcription was performed. Cells of S. mutans T8 and mutant T8-1 were grown in TSBY/CDM medium under the optimal conditions for mutacin production (20) and harvested at early stationary phase. Total RNA was isolated from both strains, and equal amounts were used for primer extension with a primer (5′-CTTCATTCAAAGAAACTACTGCGTTACTG-3′) which is complementary to codons 6 through 15 of the mutA structural gene. As shown in Fig. 3, the transcription level of mutA in mutant T8-1 was significantly reduced (about eightfold) compared with wild-type T8. RNA samples harvested from various growth phases (early log, middle log, late log, and stationary phase) confirmed these results (data not shown).

FIG. 3.

Primer extension analysis of the transcription of mutacin structural gene mutA in S. mutans T8 and mutant T8-1. Total RNA (20 μg) isolated from the wild type and the mutant grown to early stationary phase was annealed to an oligonucleotide of the mutA gene and extended with avian myeloblastosis virus reverse transcriptase. Lanes G, A, T, and C contain a dideoxy sequencing ladder carried out with the same primer. Lane 1, product from T8; lane 2, product from mutant T8-1.

Even though the mutA gene was still transcribed, albeit at a lower level, mutacin assay (4, 23) indicated that no mutacin production was detectable in mutant T8-1 (data not shown). This observation leads us to hypothesize that DGK may be involved with the regulation of mutacin production at several levels: not only at the mutA transcriptional level but also possibly at translational and posttranslational levels.

Restoring mutacin production in mutant T8-1.

To determine if mutacin production could be restored in mutant T8-1, complementation experiments were performed. The fragment (bp 537 to 1168) encoding the complete DGK was PCR amplified and cloned into vector pNoTA/T7 and then digested with BamHI and subcloned into shuttle vector pVA891 at the BamHI site. The resultant plasmid, designated pCBD5, was used to transform T8-1 by methods previously described (27). Twelve erythromycin- and tetracycline-resistant transformants were randomly picked and checked for mutacin production. All transformants produced mutacin, indicating that mutacin production in the mutant was restored by complementation with the complete dgk gene (data not shown). PCR analysis of these mutacin-positive T8-1 transformants indicated that transposon Tn916 was still in the same position relative to the downstream G-protein gene and dgk as in the T8-1 parent (data not shown). This finding further supports the assumption that DGK is involved in the regulation of mutacin II expression. Mutacin production, however, did not increase when the whole operon was provided in trans in plasmid pVA838 compared to the complemented strain containing only the dgk gene.

Previous studies have shown that an intact, full-length copy of the dgk gene is required for normal responses to various environmental stresses (pH, temperature, and osmotic pressure) in S. mutans GS5Tn1 (35). Strain T8-1 showed a similar inability to respond to changes in pH (data not shown). These findings thus link the expression of the lantibiotic mutacin II with stress response in this species. A connection between environmental stress response and bacteriocin production was reported for S. mutans JH1005 (10), in which the insertional inactivation of the fhs gene resulted in acid sensitivity and defects in production of bacteriocin JH1005. Production of some bacteriocins (e.g., colicins) in gram-negative bacteria appears to be part of the stress response upon damage of DNA by various means, including UV light. Among other genes, lexA and recA are required for the production of these bacteriocins and other extracellular proteins in E. coli and Serratia marcescens (1, 8). However, mutacin production in S. mutans appears to be recA independent (26a).

DGK may play a role in signal transduction in both eukaryotic and prokaryotic cells (13, 31, 35). Sequence alignment (30) of five prokaryotic DGKs (from B. subtilis, E. coli, Pseudomonas denitrificans, Rhizobium meliloti, and S. mutans) showed that the DGK from S. mutans has a long unique C-terminal tail after the end of the third membrane-spanning helix. The residues found to be essential for the activity of the enzyme in E. coli residing in the second cytoplasmic domain (33) are also present in S. mutans DGK. The deduced amino acid sequence of the truncated DGK in mutant T8-1 showed the absence of this unique C terminus in an otherwise unchanged protein. It is thus possible that this unique C terminus is involved in signal transduction.

In summary, by using transposonal mutagenesis, we identified another gene (dgk) essential for mutacin II production in S. mutans T8. To our knowledge, this is the first report of such a relationship among the lantibiotic-producing bacteria. Additional studies are planned to determine the functional relationship of DGK to mutacin expression and its role as a possible signal transducer.

Nucleotide sequence accession number.

The nucleotide sequence of the dgk locus has been deposited in the GenBank database under accession no. AF000954.