Conserved Mutations in the Pneumococcal Bacteriocin Transporter Gene, blpA , Result in a Complex Population Consisting of Producers and Cheaters

Abstract

All fully sequenced strains of Streptococcus pneumoniae possess a version of the blp locus, which is responsible for bacteriocin production and immunity. Activation of the blp locus is stimulated by accumulation of the peptide pheromone, BlpC, following its secretion by the ABC transporter, BlpA. The blp locus is characterized by significant diversity in blpC type and in the region of the locus containing putative bacteriocin and immunity genes. In addition, the blpA gene can represent a single large open reading frame or be divided into several smaller fragments due to the presence of frameshift mutations. In this study, we use a collection of strains with blp-dependent inhibition and immunity to define the genetic changes that bring about phenotypic differences in bacteriocin production or immunity. We demonstrate that alterations in blpA, blpC, and bacteriocin/immunity content likely play an important role in competitive interactions between pneumococcal strains. Importantly, strains with a highly conserved frameshift mutation in blpA are unable to secrete bacteriocins or BlpC, but retain the ability to respond to exogenous peptide pheromone produced by cocolonizing strains, stimulating blp-mediated immunity. These “cheater” strains can only coexist with bacteriocin-producing strains that secrete their cognate BlpC and share the same immunity proteins. The variable outcome of these interactions helps to explain the heterogeneity of the blp pheromone, bacteriocin, and immunity protein content.

The HtrA Protease of Streptococcus pneumoniae Controls Density-Dependent Stimulation of the Bacteriocin blp Locus via Disruption of Pheromone Secretion

“…In order to assess the role of HtrA in strains with different pherotypes, we created a series of blp reporter strains based on the R6 laboratory strain that differed only by their blpHC sequences. These constructs were created in a derivative of the R6 laboratory strain in which the existing, nonfunctional blp locus was replaced with a functional locus derived from strain P764 (8). These strains contain a reporter plasmid insertion at the 5= end of the BIR region that results in a lacZ gene fused to the promoter driving pneumocin and immunity protein production (Fig.…”

“…In order to detect the transcriptional activity of the blp locus, we utilized an existing R6 derivative (PSD100) that contained an active blp locus from a serotype 6B strain (P639). The construction of this strain has been previously described (8). In addition to a portion of the type 6B blp locus that includes blpA downstream of the BIR, this strain contains a reporter plasmid integration at the proximal BIR promoter, resulting in a promoterless lacZ gene fused to the promoter driving pneumocin gene expression (Fig.…”

“…Pneumocins, such as the two-peptide bacteriocin pneumocin MN, have been shown to play an important role in intraspecies competition in a murine model (4). We had previously demonstrated that variations in the integrity of BlpA, the BlpC specificity of BlpH, and the specific content of bacteriocin and immunity peptides all play an important role in dictating the outcome of competition between different pneumococcal strains (8). As an example, a highly conserved 4-bp insertion in blpA can be found in nearly half of strains studied.…”

“…This insertion results in a frameshift mutation that renders the secretion apparatus nonfunctional. Strains with a nonfunctional blpA gene are unable to secrete pheromone or pneumocins but are still able to respond to exogenous pheromone due to a preserved two-component regulatory system allowing for the induction of functional immunity genes (8). These "cheater" strains were presumably selected for because they avoid the energetic cost of pheromone and pneumocin secretion.…”

An Electrostatic Interaction between BlpC and BlpH Dictates Pheromone Specificity in the Control of Bacteriocin Production and Immunity in Streptococcus pneumoniae

“…Phospho-BlpR then binds to a consensus sequence found in promoters in the blp locus and induces the upregulation of all the genes in the locus, resulting in the expression of the bacteriocins and cognate immunity proteins encoded in the bacteriocin immunity region (BIR). The BIR of each strain contains the sequences for an assortment of as many as 11 distinct bacteriocin-like peptides, with the peptides encoded by blpMN and blpIJK being experimentally implicated in interstrain inhibitory activity (12,16,17).…”