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

Association of competence and bacteriocin/β-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.

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/β-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/β-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.

Involvement of ComAB in streptocin/β-hemolysin export.

As streptocin/β-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/β-hemolysin (sth) locus.

To identify the genetic locus encoding streptocin/β-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 β-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/β-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/β-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/β-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 (Gly→Arg) specified by the G→C 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 (G→C) in sthB resulting in an amino acid change (Gly→Arg) 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/β-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.