Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

Abstract

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.

Fig. 1

A. Time-course of nlmAp-luc gene expression in strain UA140::Φ(nlmAp-luc). The experiments were repeated two times with variations of less than 20% in between experiments. Presented here is one of the experiments. Values are averages of triplicate samples. B. nlmAp-luc gene expression in planktonic and pelleted cultures over time. Experiments were repeated three times. Presented here are the average values of the three independent experiments. RLU, relative light unit. C. Confocal micrograph of strain UA140::Φ(nlmAp–gfp) cells in pelleted culture. D. Confocal micrograph of strain UA140::Φ(nlmAp–gfp) cells in planktonic culture.

Fig. 2

nlmAp-luc gene expression in various mutant backgrounds in planktonic and pelleted cultures. Luciferase activity (RLU) was normalized with cell density (OD₆₀₀) and expressed as a percentage of the wild-type level in planktonic culture, which was arbitrarily assigned as 100%. Values shown here represent the mean of at least two independent experiments performed in triplicate.

Fig. 3

Response of mutacin IV gene expression to CSP. A. Response of nlmAp-luc gene expression to different concentrations of CSP. B. Expression of nlmAp-luc gene expression in the wild-type, comE null, and comC null background in response to CSP. The experiment was repeated three times, and values are averages of the three experiments with triplicate samples of each. C. Confocal image of nlmAp–gfp gene expression in response to CSP addition. D. Confocal image of mutpA-gfp gene expression in response to CSP addition.

Fig. 4

Coordination of competence development and mutacin IV gene expression. A. Time-course of nlmAp-luc gene expression in response to CSP addition. B. Transformation frequency of the same culture as (A). S1, no CSP; S2, with CSP. Transformation frequency was calculated as the ratio of transformants versus the number of total viable cells per ml in the transforming culture. The data shown are averages of two independent experiments assayed in triplicate. Although no error bars could be shown in (B) because of the 3-D projection, the variation between experiments is less than 20%.

Fig. 5

Transformation assay of S. mutans in mixed cultures with S. gordonii/pVA838 in the absence (A) and presence (B) of CSP. Transformation frequency was calculated as in Fig. 4. Data presented here are averages of two independent experiments assayed in triplicate.

Fig. 6

Quantification of DNA release from S. gordonii/pVA838 mediated by partially purified mutacin IV. A. Inhibitory activity of the partially purified mutacin IV (crude extract) from strain UA140I⁻IV⁺ and the control extract from strain UA140I⁻IV⁻ against S. gordonii. Each spot contained 10 μl of twofold serially diluted extract (e.g. #1, undiluted; #2, twofold diluted; #3, fourfold diluted, etc.). B. Real-time PCR quantification of DNA release from S. gordonii/pVA838 treated with crude extracts from UA140I⁻IV⁺ and UA140I⁻IV⁻. The amount of DNA released upon treatment with UA140I⁻IV⁺ crude extract at 1 min was arbitrarily assigned as 100%, which was used to normalize all samples. The values shown here represent the mean of at least two independent experiments done in triplicate.

Fig. 7

Model for coordinated mutacin IV production and competence in DNA release and uptake from other streptococcal species (see text for details).