Development of competence for genetic transformation of Streptococcus mutans in a chemically defined medium

Abstract

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).

Fig 1

Design of reporter plasmids used to monitor expression of competence regulons. (A) Elements of the type II ComR cell-to-cell communication circuit proposed as the proximal regulator of sigX expression. ComR is proposed to be a direct regulator of the transcription of comS and sigX. ComS acts as a cell-cell signal, but the steps of its processing into a mature form (XIP) and its export are unknown. SigX is the proximal regulator of late competence effector genes. (Reproduced, with permission, from part of a figure in reference .) (B) Organization of the replicative plasmid pWAR304, a luxAB transcriptional reporter of expression from the sigX promoter. ori, replication origin from plasmid pWVO1 from Lactococcus lactis (11); P_sigX, fragment upstream of sigX; RepA, repA gene from pWVO1. (C) Organization of the integrative plasmid pWAR312, a luxAB transcriptional reporter of expression from the sigX promoter. attP, integrase target; attB, insertion site in the genome of UA159; int, integrase gene; ermB, rRNA methyl transferase from pAMβ1; ori, replication origin from plasmid pUC18 (18).

Fig 2

Effect of synthetic ComS_11-17 peptide on growth, sigX expression, and competence in S. mutans cultured in CDM at low density. Five parallel 22-ml cultures of MW30 growing in CDM at an OD₅₅₀ of 0.1 were treated with 0.1 μM, 1 μM, or 10 μM ComS_11-17; 1 μM (each) XIP peptides predicted for S. agalactiae 2603 (GBS), S. porcinus (POR), and S. parauberis (SPU); or vehicle alone at time zero. (A) Growth was monitored as the OD₅₅₀. (B) To determine competence at 30-min intervals, MW07 donor DNA (5 μg) was added to 500-μl samples from the cultures; after 8 min at 37°C, DNase was added, and dilutions were plated to determine Cm^r transformants after 60 min of further incubation. (C) To determine expression from the sigX promoter, triplicate 50-μl samples from each tube were exposed to decanal vapor in a 96-well plate for 2 min before determining luminescence.

Fig 3

Dose dependence of responses to ComS_11-17. (A) Reduction of the growth rate in CDM by added ComS_11-17. Twelve 6-ml cultures of UA159 were grown in CDM after dilution of an overnight CDM culture to an OD₅₅₀ of 0.012. At 155 min, ComS_11-17 was added at 0.01, 0.03, 0.1, or 1 μM, and growth was monitored for an additional 350 min. The values shown are the averages of duplicate parallel cultures. (B) Titration of competence response to ComS_11-17. Samples (1 ml) of a culture of UA159 growing in CDM at an OD₅₅₀ of 0.1 were treated with the indicated concentrations of ComS_11-17 for 60 min and then exposed to MW07 DNA at 10 μg/ml for 60 min before plating to determine Cm^r transformants. (C) Donor DNA dose-response. A culture of UA159 growing in CDM at an OD₅₅₀ of 0.1 was treated with 10 μM ComS_11-17 for 60 min. Samples were then exposed for 20 min to the indicated amounts of MW07 DNA at 37°C, diluted, and plated to determine Cm^r transformants. (D) Reduced sensitivity to ComS_11-17 in rich media. A culture of UA159 growing in CDM at an OD₅₅₀ of 0.1 was treated with the indicated amounts of ComS_11-17 for 60 min after supplementation with THB as indicated. Finally, competence was assayed by a further 30-min incubation with 0.1 μg/ml MW04 DNA before plating to determine Spc^r transformants. A parallel culture of UA159 in THB was treated at an OD₅₅₀ of 0.1 in the same way.

Fig 4

Bioassay of ComS_11-17. (A) Kinetics of response of the comS mutant to ComS_11-17. Samples (250 μl) of MW17 (ΔcomS P_sigX-luxAB) growing in CDM at an OD₅₅₀ of 0.4 were mixed with 250-μl volumes of CDM containing ComS_11-17 at twice the indicated final concentrations. During incubation at 37°C, 60-μl portions were removed at the indicated times for exposure to decanal and determination of luminescence. (B) ComS_11-17 bioassay standard curve. The titration curve was constructed using the MW17 luminescence values at 80 min from the experiment in panel A.

Fig 5

Endogenous competence development in CDM. (A) Development of competence, expression from PsigX, and pheromone secretion by strain MW30 (P_sigX-luxAB) during growth in CDM in parallel closed culture tubes. The tubes were opened on reaching the indicated OD₅₅₀ values, and their contents were distributed for three assays. MW07 donor DNA (0.5 μg) was added to one 500-μl sample from each culture; after 15 min at 37°C, DNase was added and dilutions were plated for Cm^r transformants after 60 min of further incubation. Triplicate 150-μl samples from each tube were exposed to decanal vapor in a 96-well plate for 2 min before determining luminescence. A 4-ml filtrate of the remainder (0.2-μm filters) was prepared for determination of XIP as comS-complementing activity (ComS_11-17 equivalents) by bioassay, as described in Materials and Methods. (B) Growth kinetics of cultures assayed for panel A.