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Journal of Bacteriology, February 2007, p. 1468-1472, Vol. 189, No. 4
0021-9193/07/$08.00+0     doi:10.1128/JB.01174-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Competence-Dependent Bacteriocin Production by Streptococcus gordonii DL1 (Challis)

Nicholas C. K. Heng,^1,2* John R. Tagg,² and Geoffrey R. Tompkins^1*

Department of Oral Sciences,¹ Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand²

Received 30 July 2006/ Accepted 19 September 2006

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The production of streptocins STH₁ and STH₂ by Streptococcus gordonii DL1 (Challis) is directly controlled by the competence regulon, which requires intact comR and comAB loci. The streptocin (sth) locus comprises two functional genes, sthA and sthB. Whereas STH₁ activity requires sthA alone, STH₂ activity depends on both genes.

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In the 1970s, Schlegel and Slade (20-22) reported the production of streptocin STH₁, a proteinaceous antimicrobial agent (bacteriocin), by the naturally transformable Streptococcus gordonii (formerly Streptococcus sanguis) strain Challis (also known as DL1 and NCTC 7868). Streptocin STH₁ biosynthesis coincided with the development of competence for genetic transformation (20, 21), a relationship supported by later studies (8, 23). The inhibitory spectrum of streptocin STH₁ was initially reported to include other S. gordonii strains (e.g., strains Wicky and C219) and select strains of Streptococcus mitis and Streptococcus oralis (8, 23). However, we have observed that the bacteriocin activity targeting S. mitis and S. oralis appears to be linked to the beta-hemolytic phenotype of strain Challis because S. gordonii OB164 (originally designated DL1-Challis), a non-beta-hemolytic strain indistinguishable from DL1 biochemically (API 20 Strep; bioMérieux, France) and genotypically (18), exhibited inhibitory activity only against S. gordonii isolates. This suggested that strain DL1 produces two bacteriocins: the previously described STH₁ (20) and the newly designated STH₂, a bacteriocin that targets certain non-S. gordonii strains and is associated with the beta-hemolytic phenotype. Based on our speculation that genetic competence and bacteriocin production may be functionally related in strain DL1 (23), the aims of the present study were to determine whether bacteriocin biosynthesis is genetically associated with competence development and to define the locus encoding the inhibitory/hemolytic agents.

(Parts of this work were presented at the 7th ASM Conference on Streptococcal Genetics, Saint Malo, France [7a].)

Association of competence and bacteriocin/ß-hemolysin production. Competence development in S. gordonii is analogous to competence development in Streptococcus pneumoniae, occurring in two distinct stages, early and late (10, 13). The early stage involves a quorum-sensing signal transduction circuit comprising a secreted competence-stimulating peptide (CSP), its cell surface receptor ComD (a histidine kinase), and ComE, the primary transcriptional regulator of competence (10, 13). ComE, when activated by ComD, upregulates the comCDE (signal transduction), comAB (CSP secretion), and comX operons (13). ComX, an alternative sigma factor, connects the early and late stages by activating expression of the late competence operons that are responsible for DNA uptake and processing (17). Several key components of the S. gordonii competence regulon have been identified, including comCDE, comAB, comYA (DNA uptake), and duplicate comR (comX homologue) loci (6, 7, 15, 16).

In this study, early (comC, comE, comA, comB, and comR) and late (comYA) competence genes of strain DL1 were individually inactivated by insertion of the erythromycin resistance determinant ermAM (2) via double-crossover homologous recombination. The second copy of comR was disrupted with the kanamycin resistance marker aphA3 (7, 24). The relevant mutagenic plasmids and derived strains are listed in Table 1. A complete list of oligonucleotide primers and their sequences is available upon request. Insertion of ermAM was always in the same transcriptional orientation as the gene of interest, and the absence of a transcription terminator downstream of ermAM was expected to preclude any polar effects. All desired mutations were confirmed by the sizes of PCR amplicons generated using appropriate primer combinations and by sequencing of PCR products. Competence (transformability) and streptocin production were assessed as described previously (23), except that the assays were standardized to the growth phase of the cultures (optical density at 600 nm, 0.1 to 0.15) instead of time. Hemolysis was assessed by incubating bacteria under anaerobic conditions on horse blood agar. Under these growth conditions, beta-hemolytic and nonhemolytic colonies are readily distinguishable; Alpha-hemolysis is not observed.

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TABLE 1. Bacterial strains and plasmids used in this study

As shown in Table 2, individual inactivation of comC, comE, comA, and comB resulted in nontransformable, nonbacteriocinogenic, nonhemolytic derivatives. In contrast, the comYA-defective mutant (NHG306) was not transformable but exhibited both bacteriocin and beta-hemolytic activities (Table 2). To clarify whether bacteriocin/ß-hemolysin production is regulated at the early or late stage of competence, single- and double-knockout mutants (strains NHG308, NHG309, and NHG310) with mutations in the competence-specific sigma factor structural gene (comR) were derived. Transformability, bacteriocinogeny, and beta-hemolysis were eliminated only in the comR double-knockout mutant NHG310, and all three phenotypes were restored when comR was supplied in trans either on pNCKH311 (comR1) or on pNCKH312 (comR2) (Table 2). The single-knockout comR mutants, NHG308 and NHG309, exhibited essentially wild-type characteristics, presumably due to the compensation effected by the other intact comR allele (Table 2). Dependence on ComR, therefore, indicated that streptocin/ß-hemolysin production is a late competence phenomenon, and bacteriocin production by the comYA-defective mutant confirmed the independence from the DNA uptake and transformation mechanisms.

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TABLE 2. Competence and bacteriocinogenic and beta-hemolytic properties of S. gordonii strains

Involvement of ComAB in streptocin/ß-hemolysin export. As streptocin/ß-hemolysin biogenesis is a late competence event, we anticipated that exogenously supplied synthetic CSP would bypass certain elements of the early competence (comABCDE) circuit, restoring wild-type characteristics to the comC, comA, and comB knockout mutants but not to the comE and double-knockout comR mutants. However, inhibitory activity was restored only to the comC mutant (Table 2), implicating the ABC transporter complex ComAB in streptocin export. ComAB secretes mature CSP following cleavage of the precursor peptide ComC at a specific Gly-Gly motif (10), and ComAB-like transporters (e.g., NlmTE in Streptococcus mutans [4]) are common exit portals for nonmodified (i.e., class II) Gly-Gly-containing peptide bacteriocins (3, 5). Furthermore, a BLAST (1) search of the S. gordonii Challis genome sequence did not reveal an alternative ABC transport system.

Identification of the streptocin/ß-hemolysin (sth) locus. To identify the genetic locus encoding streptocin/ß-hemolysin, we exploited the transformable nature of the beta-hemolytic phenotype (9). Strain OB164 (nonhemolytic) was transformed with a DL1 subgenomic library cloned into pFX3 (27), and chloramphenicol-resistant transformants were screened for beta-hemolysis. Interestingly, streptocins STH₁ and STH₂ were both produced by several beta-hemolytic transformants tested (data not shown), confirming that the bacteriocin and ß-hemolysin structural genes are closely linked. Strain NHG323, which carries pNCKH323, was selected for further analysis.

End sequencing of the pNCKH323 insert followed by BLAST-assisted searches of the S. gordonii genome sequence revealed the organization of the chromosomal region responsible for beta-hemolysis (Fig. 1A). The streptocin/ß-hemolysin (sth) locus consists of three open reading frames (ORFs) flanked by nanE (encoding N-acetylmannosamine-6-phosphate epimerase) and adhE (encoding alcohol-acetaldehyde dehydrogenase) (Fig. 1A). An 8-bp sequence, 5'-TGCGAATA-3', located 116 nucleotides upstream of ORF1, closely resembles a ComX recognition/binding site or cin box (5'-TACGAATA-3') (13, 17). As the variant sequence serves as the cin box for the late competence cgl operon in S. pneumoniae (14), and since streptocin/ß-hemolysin production is ComR dependent, we anticipated that this octanucleotide motif functions as the cin box for the sth locus. This conclusion is supported by the results of recent transcriptomic analyses of CSP-induced loci in S. gordonii Challis (25a), in which expression of sthA and sthB was detected 15 min after CSP addition, corresponding to a late competence response (M. Vickerman, personal communication).

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FIG. 1. (A) Genomic organization of the sth locus in S. gordonii DL1 (Challis). The solid arrows represent genes that were completely sequenced in this study. The putative ComR recognition site (cin box; 5'-TGCGAATA-3') and a predicted rho-independent transcription terminator are represented by a striped box and a lollipop symbol, respectively. CHP, conserved hypothetical protein; nanE, N-acetylmannosamine-6-phosphate epimerase; sthA and sthB, genes encoding prepeptides of streptocin/ß-hemolysin; adhE, gene encoding alcohol-acetaldehyde dehydrogenase. (B) Deduced peptides encoded by the three ORFs of the sth locus. The site adjacent to the Gly-Gly motif, where cleavage is expected to occur during export, is indicated by the inverted arrow. The amino acid change (GlyArg) specified by the GC mutation in the sthB gene of strain OB164 is also indicated. (C) Synergism between the translated products of sthA and sthB generating beta-hemolysis. Twenty microliters each of diluted (1:100) overnight Todd-Hewitt broth cultures of strains NHG327 (sthA+) and NHG326 (sthB+) were spotted 3 mm apart on horse blood agar and incubated at 37°C under anaerobic conditions for 18 h.

ORF1, ORF2, and ORF3 encode polypeptides having 46, 57, and 53 amino acid residues, respectively (Fig. 1B). All three peptides contain putative signal peptides with Gly-Gly motifs, a prerequisite for processing and secretion by peptide-exporting ABC-type transport systems (5), further supporting the hypothesis that ComAB is involved in streptocin export. Whereas the peptide encoded by ORF1 exhibits no homology to any known protein, the translation products of ORF2 and ORF3 exhibit significant similarity (58% to 81%) to two peptides having unknown functions (Lmes02001476 and Lmes02001478, respectively) from Leuconostoc mesenteroides ATCC 8293 (GenBank accession no. NZ_AABH02000035).

In order to determine its function in streptocin or beta-hemolytic activity, each ORF in the sth locus was insertionally inactivated with ermAM. Disruption of ORF1 had no appreciable effect on either bacteriocinogeny or beta-hemolysis (Table 2), and the function of the ORF1 gene product, if any, remains to be determined. In contrast, inactivation of either ORF2 (renamed sthA) or ORF3 (sthB) eliminated both streptocin STH₂ activity and beta-hemolysis (Fig. 1C and Table 2), confirming the linkage of these two phenotypes. Strain NHG327 (sthA⁺ sthB::ermAM) exhibited streptocin STH₁ activity, indicating that while SthA alone effects STH₁ activity, STH₂ activity or beta-hemolysis may depend on synergism between the SthA and SthB peptides. To confirm this, strains NHG326 and NHG327 (neither of which is beta-hemolytic individually) were grown in proximity on horse blood agar, which resulted in a zone of beta-hemolysis between the two cultures (Fig. 1C). Similarly, a 1:1 mixture of culture supernatants from NHG326 and NHG327 inhibited the growth of the STH₂ indicator strain S. mitis I18 (data not shown), whereas neither supernatant alone was inhibitory (Table 2).

Sequence analysis of the sth locus in strain OB164 revealed a single nucleotide polymorphism (GC) in sthB resulting in an amino acid change (GlyArg) in the translational product (Fig. 1B). This substitution alters the hydrophobicity profile of the exported peptide (data not shown), probably accounting for its loss of function. Indeed, strain OB164 could substitute for strain NHG327 (sthB mutant) in the hemolysis complementation experiment shown in Fig. 1C.

Concluding remarks. In this study, genetic dissection confirmed that bacteriocin/ß-hemolysin biogenesis in S. gordonii strain DL1 is directly controlled by the competence regulon. Dependence on a functional comR gene and detection of a putative cin box consign expression of the sth locus to the late competence phase. Furthermore, the inability of synthetic CSP to restore bacteriocin production in the comAB knockout mutants and the presence of signal peptides with Gly-Gly motifs in SthA and SthB indicate that both prepeptides are probably processed and exported by ComAB, yielding SthA* and SthB*, respectively. Whereas SthA* independently manifests as streptocin STH₁, both SthA* and SthB* are required to effect streptocin STH₂/beta-hemolytic activity.

The concept that bacteriocin production might influence transformation was first proposed by Jyssum and Allunans (11), who reported that coculture of competent, bacteriocinogenic isolates of Neisseria with inhibitor-sensitive strains resulted in transformation of the former with antibiotic resistance markers derived from the latter. Similarly, Kreth et al. (12) reported that mutacin IV, a bacteriocin secreted by some strains of S. mutans, lyses target bacteria (including S. gordonii) and releases DNA which can then transform the bacteriocin producer. Wang and Kuramitsu (26) have also demonstrated that production of mutacin Smb by S. mutans strains BM71 and GS5 requires a functional CSP-mediated ComDE signal transduction system. Thus, competence-associated bacteriocin production by oral bacteria could enhance transformation either (i) by killing and lysing related strains and thus releasing potentially useful genetic material with which the bacteriocinogenic strain self-transforms or (ii) by eliminating other competent strains competing for limited DNA in the oral cavity (23). As streptocins STH₁ and STH₂ do not appear to be bacteriolytic (22, 23), it seems that the latter function is more plausible. Perhaps more significantly, mutacin IV is an early competence product (12, 25), which may be consistent with its function of releasing DNA prior to activation of the DNA uptake machinery, whereas the late competence biosynthesis of STH₁ and STH₂ by strain Challis may reflect anticompetitor functions concomitant with DNA uptake. Strain Challis produces a protease, challisin, that degrades S. mutans CSP, thereby interfering with CSP-mediated mutacin biogenesis (26). This suggests that strain Challis possesses both biological weaponry (streptocins) and defensive capability (challisin).

Although the ecological functions of bacteriocins have rarely been elucidated (19), previous findings (11, 12) and the results of the present investigation support the concept that some bacteriocins facilitate genetic transformation (23). At an applied level, the enhancement of horizontal gene transfer by bacteriocin expression should be considered when the potential therapeutic and prophylactic uses of bacteriocinogenic bacteria are assessed.

Nucleotide sequence accession number. The nucleotide sequence of the sth locus has been deposited in the GenBank database under accession number AY858646.

 
This study was supported by the Marsden Fund (Royal Society of New Zealand) and by the New Zealand Lottery Grants Board.

We are grateful to The Institute for Genomic Research, which provided preliminary S. gordonii Challis NCTC 7868 genome sequence data, to D. Harding (Centre for Separation Science, Massey University, New Zealand) for synthesis of CSP, and to M. Vickerman for sharing prepublication S. gordonii microarray data. We also thank R. Jack for a critique of the manuscript.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. Phone for N. C. K. Heng: 64 3 479-5155. Fax: 64 3 479-8540. E-mail: nicholas.heng{at}stonebow.otago.ac.nz. Phone for G. R. Tompkins: 64 3 479-5471. Fax: 64 3 479-7078. E-mail: geoffrey.tompkins{at}stonebow.otago.ac.nz.

Published ahead of print on 29 September 2006.

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1
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
2
2. Brehm, J., G. Salmond, and N. Minton. 1987. Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAMß1. Nucleic Acids Res. 15:3177.[Free Full Text]
3
3. Eijsink, V. G. H., L. Axelsson, D. B. Diep, L. S. Havarstein, H. Holo, and I. F. Nes. 2002. Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Leeuwenhoek 81:639-654.[CrossRef][Medline]
4
4. Hale, J. D. F., N. C. K. Heng, R. W. Jack, and J. R. Tagg. 2005. Identification of nlmTE, the locus encoding the ABC transport system required for export of nonlantibiotic mutacins in Streptococcus mutans. J. Bacteriol. 187:5036-5039.[Abstract/Free Full Text]
5
5. Håvarstein, L. S., D. B. Diep, and I. F. Nes. 1995. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol. Microbiol. 16:229-240.[Medline]
6
6. Håvarstein, L. S., P. Gaustad, I. F. Nes, and D. A. Morrison. 1996. Identification of the streptococcal competence pheromone receptor. Mol. Microbiol. 21:863-869.[CrossRef][Medline]
7
7. Heng, N. C. K., J. R. Tagg, and G. R. Tompkins. 2006. Identification and characterization of the loci encoding the competence-associated alternative sigma factor of Streptococcus gordonii. FEMS Microbiol. Lett. 259:27-34.[CrossRef][Medline]
7
8. Heng, N. C. K., J. R. Tagg, and G. R. Tompkins. 2006. 7th ASM Conference on Streptococcal Genetics, Saint Malo, France, abstr. A96.
8
9. Ito, T. 1982. Group H streptococcal bacteriocins having no relation to bacterial transformation. Microbiol. Immunol. 26:93-105.[Medline]
9
10. Ito, T., T. Hirano, T. Tomura, and M. Yoshioka. 1973. Transformability of group H Streptococcus Challis. II. Transformation of hemolytic activity and competence-provoking factor nonproducibility. Jpn. J. Microbiol. 17:439-444.[Medline]
10
11. Jenkinson, H. F., and M. M. Vickerman. 2006. Genetics of sanguinis group streptococci, p. 347-355. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood (ed.), Gram-positive pathogens, 2nd ed. American Society for Microbiology, Washington, DC.
11
12. Jyssum, K., and J. Allunans. 1983. Expression of bacteriocin-like activity in batch cultures of Neisseria meningitidis and consequences for genetic recombination. Acta. Pathol. Microbiol. Immunol. Scand. Sect. B 91:107-111.[Medline]
12
13. Kreth, J., J. Merritt, W. Shi, and F. Qi. 2005. Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species. Mol. Microbiol. 57:392-404.[CrossRef][Medline]
13
14. Lacks, S. A. 2004. Transformation, p. 89-115. In E. I. Tuomanen, T. J. Mitchell, D. A. Morrison, and B. G. Spratt (ed.), The pneumococcus. American Society for Microbiology, Washington, DC.
14
15. Lacks, S. A., S. Ayalew, A. de la Campa, and B. Greenberg. 2000. Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system. Mol. Microbiol. 35:1089-1098.[CrossRef][Medline]
15
16. Lunsford, R. D., and A. G. Roble. 1997. comYA, a gene similar to comGA of Bacillus subtilis, is essential for competence-factor-dependent DNA transformation in Streptococcus gordonii. J. Bacteriol. 179:3122-3126.[Abstract/Free Full Text]
16
17. Lunsford, R. D., and J. London. 1996. Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain Wicky. J. Bacteriol. 178:5831-5835.[Abstract/Free Full Text]
17
18. Luo, P., H. Li, and D. A. Morrison. 2003. ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae. Mol. Microbiol. 50:623-633.[CrossRef][Medline]
18
19. Rahimi, M., N. C. K. Heng, J. A. Kieser, and G. R. Tompkins. 2005. Genotypic comparison of bacteria recovered from human bite marks and teeth using arbitrarily primed PCR. J. Appl. Microbiol. 99:1265-1270.[CrossRef][Medline]
19
20. Riley, M. A., and J. E. Wertz. 2002. Bacteriocins: evolution, ecology, and application. Annu. Rev. Microbiol. 56:117-137.[CrossRef][Medline]
20
21. Schlegel, R., and H. D. Slade. 1972. Bacteriocin production by transformable group H streptococci. J. Bacteriol. 112:824-829.[Abstract/Free Full Text]
21
22. Schlegel, R., and H. D. Slade. 1973. Properties of a Streptococcus sanguis (group H) bacteriocin and its separation from the competence factor of transformation. J. Bacteriol. 115:655-661.[Abstract/Free Full Text]
22
23. Schlegel, R., and H. D. Slade. 1974. Alteration of macromolecular synthesis and membrane permeability by a Streptococcus sanguis bacteriocin. J. Gen. Microbiol. 81:275-277.[Abstract/Free Full Text]
23
24. Tompkins, G. R., M. A. Peavey, K. R. Birchmeier, and J. R. Tagg. 1997. Bacteriocin production and sensitivity among coaggregating and noncoaggregating oral streptococci. Oral Microbiol. Immunol. 12:98-105.[Medline]
24
25. Trieu-Cuot, P., and P. Courvalin. 1983. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5'-aminoglycoside phosphotransferase type III. Gene 23:331-341.[CrossRef][Medline]
25
26. van der Ploeg, J. R. 2005. Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence. J. Bacteriol. 187:3980-3989.[Abstract/Free Full Text]
25
27. Vickerman, M. M., S. Iobst, A. M. Jesionowski, and S. R. Gill. 2006. 7th ASM Conference on Streptococcal Genetics, Saint Malo, France, abstr. A224.
26
28. Wang, B.-Y., and H. K. Kuramitsu. 2005. Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii. Appl. Environ. Microbiol. 71:354-362.[Abstract/Free Full Text]
27
29. Xu, F., L. E. Pearce, and P.-L. Yu. 1991. Construction of a family of lactococcal vectors for gene cloning and translational fusions. FEMS Microbiol. Lett. 77:55-60.[CrossRef]
28
30. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]

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Journal of Bacteriology, February 2007, p. 1468-1472, Vol. 189, No. 4
0021-9193/07/$08.00+0     doi:10.1128/JB.01174-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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