Coordinated Bacteriocin Expression and Competence in Streptococcus pneumoniae Contributes to Genetic Adaptation through Neighbor Predation

Abstract

Streptococcus pneumoniae (pneumococcus) has remained a persistent cause of invasive and mucosal disease in humans despite the widespread use of antibiotics and vaccines. The resilience of this organism is due to its capacity for adaptation through the uptake and incorporation of new genetic material from the surrounding microbial community. DNA uptake and recombination is controlled by a tightly regulated quorum sensing system that is triggered by the extracellular accumulation of competence stimulating peptide (CSP). In this study, we demonstrate that CSP can stimulate the production of a diverse array of blp bacteriocins. This cross stimulation occurs through increased production and secretion of the bacteriocin pheromone, BlpC, and requires a functional competence regulatory system. We show that a highly conserved motif in the promoter of the operon encoding BlpC and its transporter mediates the upregulation by CSP. The accumulation of BlpC following CSP stimulation results in augmented activation of the entire blp locus. Using biofilm-grown organisms as a model for competition and genetic exchange on the mucosal surface, we demonstrate that DNA exchange is enhanced by bacteriocin secretion suggesting that co-stimulation of bacteriocins with competence provides an adaptive advantage. The blp and com regulatory pathways are believed to have diverged and specialized in a remote ancestor of pneumococcus. Despite this, the two systems have maintained a regulatory connection that promotes competition and adaptation by targeting for lysis a wide array of potential competitors while simultaneously providing the means for incorporation of their DNA.

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.

Fig 2. CSP induction of BIR transcription is dependent on ComE, BlpH and BlpC but not ComA.

BIR _p1 -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 BIR _p1 -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), blpC _R6 H _6A 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 (BIR₁₆₄ ΔcomAB), PSD305 (BIR₁₆₄ ΔcomE) and the pneumocin non-producer, PSD299 (BIR_D39).

Fig 3. CSP induces production and secretion of BlpC.

(A) BlpC_R6 dose response in wt PSD100 (blue), ΔcomAB PSD140 (red), and ΔcomE PSD141 (green) version of BIR _p1 -lacZ reporter strains. (B) BlpC_R6 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 BlpC_FLAG strain (PSD125) after treatment with CSP or BlpC_R6. Equal amounts of protein were loaded into each lane. (D) BlpC_R6 secretion assay using chimeric BlpC_R6/H_6A (BlpC_R6 secretor, BlpC_6A responsive) BIR:lacZ reporter strains: PSD118 (blue), PSD142 (ΔblpA, purple), and PSD143 (ΔcomAB, red). Upper: BIR _p1 -lacZ induction of chimera strains 30 minutes after peptide addition. Lower: BlpC_R6 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 blpAp _G-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) BlpC_R6 dose response assay using BIR _p1 -lacZ reporter strains: PSD100 (Wt, blue circle), and PSD200 (blpAp _G-90A, orange triangle). (C) CSP induction assay using BIR _p1 -lacZ reporters, PSD100 and single nucleotide mutant at blpA promoter PSD200 (blpAp _G-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 BlpC_FLAG strains PSD125 (Wt) and PSD225 (blpAp _G-90A) after treatment with increasing concentrations of BlpC_R6. Unprocessed BlpC_R6 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 BlpC_FLAG strains PSD125 (Wt) and PSD225 (blpAp _G-90A) after treatment with CSP or BlpC_R6. Unprocessed BlpC_FLAG is denoted as pre-BlpC. (F) Inhibitory overlay assay using D39 pneumocin producing strains, PSD300 (BIR_P164), PSD303 (BIR_P164 blpAp _G-90A), and non-producing PSD299 (BIR_D39) strain. (G) Response to CSP induction in D39 background BIR _p2 -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 (blpAp _G-90A). Gray dashed lines denote the cellular growth.

Fig 5. ComA can process and secrete BlpC independently of BlpA.

(A-C) BIR _P1 -lacZ transcriptional response in wildtype (blue circles), blpA _FS (purple octagon) and blpA _FS/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 BIR _P1 -lacZ induction in competence permissive (B) and competence non-permissive C+Y media (C). (D) Western blot of cell lysate of BlpC_FLAG expressing strains in ΔhtrA background: PSD127 (Wt), PSD131 (ΔblpA), PSD146 (ΔcomAB) and PSD147 (ΔblpA/comAB) after treatment with CSP or BlpC_R6 or both BlpC_R6/CSP. Preprocessed BlpC_FLAG 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 BlpC_FLAG of the total BlpC_FLAG 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 (BIR₁₆₄ blpAp _G-90A, orange triangle), and non-producer control PSD299 (BIR_D39, 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.