Fig 1.
Schematic model of the genetic organization and regulation of the Com and Blp systems.
Genes are labeled with colored arrows, colors used match the gene products. Red arrows demonstrate regulatory pathways, black arrows represent secretion pathways. CSP is processed and secreted out of the cell by the ComAB transporter complex. When sufficient local concentrations of CSP accumulate, CSP binding to the receptor ComD activates ComE, a DNA binding protein. ComE then upregulates genes in the competence regulon either directly through binding to promoters or indirectly through induction of an alternative sigma factor. ComE upregulated genes include the genes encoding fratricide effectors and their associated immunity proteins. Blp bacteriocin expression is controlled by the local accumulation of BlpC after secretion and processing by the BlpAB complex. BlpC binds to and activates the two component system, BlpHR. Activated BlpR upregulates the genes in the blp locus including genes in the BIR that encode bacteriocins (pneumocins) and immunity proteins. Pneumocins can target neighboring cells that do not produce Blp specific immunity. In this work we provide evidence that activated ComE can upregulate the production of BlpABC through binding to and upregulating the blpABC promoter. In addition, we show that ComAB can serve as an alternative transporter for BlpC which may play a particularly important role in the absence of a functional BlpAB.
Table 1.
Strain, plasmids, primers and peptides.
Fig 2.
CSP induction of BIR transcription is dependent on ComE, BlpH and BlpC but not ComA.
BIRp1-lacZ reporters or D39 derivatives expressing inhibitory pneumocins were used to assess the response of the blp locus to competence induction. (A) Diagrammatic representation of the BIRp1-lacZ reporter fusion. This strain carries an integrated reporter plasmid in which the 5’ most BIR promoter (P1) is driving lacZ. A second BIR promoter is noted as P2. Integrated plasmid sequences are shown as white arrows. The gene designations are shown below the arrows. Derivatives of this reporter fusion were used to assay for BIR response to CSP induction in THY (B-G) or C+Y (H-I), Open symbols represent samples that were not induced with CSP, closed symbols represent cultures that were induced with 200 ng/ml CSP at time 0. Gray dashed lines denote the cellular growth. Comparison of BIR transcriptional response to CSP addition in Wt PSD100 (B), ΔcomAB PSD140(C), ΔcomE PSD141(D), ΔblpC PSD101(E), blpCR6H6A chimera PSD118(F) or ΔblpH PSD119(G) strains. Comparison of Wt, ComAB and ComE deficient reporters in natural BIR induction in competence permissive C+Y pH 8 (H) or competence non-permissive C+Y pH 6.8 (I). (J) Results of an inhibitory overlay assay using D39 pneumocin producing strains PSD300 (BIR164), PSD304 (BIR164ΔcomAB), PSD305 (BIR164ΔcomE) and the pneumocin non-producer, PSD299 (BIRD39).
Fig 3.
CSP induces production and secretion of BlpC.
(A) BlpCR6 dose response in wt PSD100 (blue), ΔcomAB PSD140 (red), and ΔcomE PSD141 (green) version of BIRp1-lacZ reporter strains. (B) BlpCR6 dose response in a ΔblpC reporter strain PSD101 (purple open) and following 10 minutes of CSP pretreatment (purple closed). (C) Western blot of cell lysate of BlpCFLAG strain (PSD125) after treatment with CSP or BlpCR6. Equal amounts of protein were loaded into each lane. (D) BlpCR6 secretion assay using chimeric BlpCR6/H6A (BlpCR6 secretor, BlpC6A responsive) BIR:lacZ reporter strains: PSD118 (blue), PSD142 (ΔblpA, purple), and PSD143 (ΔcomAB, red). Upper: BIRp1-lacZ induction of chimera strains 30 minutes after peptide addition. Lower: BlpCR6 concentration secreted into supernatant 30 minutes after induction. P values as noted were determined by student T-test.
Fig 4.
ComE-like binding site in the blpABC promoter is required for CSP induction of BlpC secretion.
(A) Sequence analysis of blp regulated promoters. Consensus sequences that support ComE or BlpR binding are shown on the top of the alignment with conserved nucleotides as capital letters. Alignment of identified BlpR binding sites from the TIGR4 blp locus promoters was adopted from [32]. A guanine in the blpA promoter binding site at -90 from the start codon is labeled with an arrow and represents the only BlpR binding site within the blp locus with this change. The qrsA promoter also has a guanine at this position; this promoter is known to be regulated by both ComE and BlpR. Gene designations identify the first gene in the operon that is controlled by the promoter, P1 and P2 refer to the BIR promoters noted in Fig 2A. The created blpApG-90A sequence is shown with the nucleotide change in orange. Shared nucleotides between the two consensus sequences and the listed binding sites are highlighted in yellow. Nucleotides that differ between the two are highlighted in either green (ComE) or blue (BlpR). (B) BlpCR6 dose response assay using BIRp1-lacZ reporter strains: PSD100 (Wt, blue circle), and PSD200 (blpApG-90A, orange triangle). (C) CSP induction assay using BIRp1-lacZ reporters, PSD100 and single nucleotide mutant at blpA promoter PSD200 (blpApG-90A). CSP induced fold change in BIR transcription normalized to uninduced samples is shown. Gray dashed lines denote the cellular growth. (D) Western blot of cell lysates of BlpCFLAG strains PSD125 (Wt) and PSD225 (blpApG-90A) after treatment with increasing concentrations of BlpCR6. Unprocessed BlpCR6 is denoted as pre-BlpC. Fold change compared with wildtype levels for each concentration is shown after values were normalized with pneumolysin (Ply) loading control. (E) Western blot of cell lysates of BlpCFLAG strains PSD125 (Wt) and PSD225 (blpApG-90A) after treatment with CSP or BlpCR6. Unprocessed BlpCFLAG is denoted as pre-BlpC. (F) Inhibitory overlay assay using D39 pneumocin producing strains, PSD300 (BIRP164), PSD303 (BIRP164blpApG-90A), and non-producing PSD299 (BIRD39) strain. (G) Response to CSP induction in D39 background BIRp2-lacZ reporter strains. CSP induced fold change in BIR transcription normalized to uninduced samples is shown for PSD306 (Wt) and single nucleotide mutant at blpA promoter PSD307 (blpApG-90A). Gray dashed lines denote the cellular growth.
Fig 5.
ComA can process and secrete BlpC independently of BlpA.
(A-C) BIRP1-lacZ transcriptional response in wildtype (blue circles), blpAFS (purple octagon) and blpAFS/comAB (red squares) mutant reporters during growth in broth. Closed symbols denote CSP addition, open symbols denote no peptide added. Cellular growth is shown as grey lines. (A) Response to CSP added at time 0 in THY. (B-C) Natural BIRP1-lacZ induction in competence permissive (B) and competence non-permissive C+Y media (C). (D) Western blot of cell lysate of BlpCFLAG expressing strains in ΔhtrA background: PSD127 (Wt), PSD131 (ΔblpA), PSD146 (ΔcomAB) and PSD147 (ΔblpA/comAB) after treatment with CSP or BlpCR6 or both BlpCR6/CSP. Preprocessed BlpCFLAG is denoted as pre-BlpC, the processed form is BlpC. Anti-pneumolysin (Ply) antibody was used as a loading control. Densitometry analysis was performed to determine % processed BlpCFLAG of the total BlpCFLAG detected in each strain.
Fig 6.
Pneumocin production promotes competition and DNA exchange in pneumococcal biofilms.
(A) Biomass of biofilms when inoculated with single competitor strains: pneumocin producers PSD300 (Wt, BIR164, blue circle) or PSD303 (BIR164blpApG-90A, orange triangle), and non-producer control PSD299 (BIRD39, blue square). (B-C) PSD300, PSD303 or PSD299 were co-inoculated with sensitive strain, D39x at ratio of 1:10 to form biofilm. Three day old biofilms were disrupted and plated on single selective media to determine (B) competition, and dual selective media to examine (C) transformation efficiency. Two-tailed Mann-Whitney tests were performed to determine statistical significance. * denotes p<0.05, *** denotes p<0.001.