The introduction of the conjugate seven-valent pneumococcal vaccine has led to the replacement of vaccine serotypes with nonvaccine serotypes of Streptococcus pneumoniae. This observation implies that intraspecies competition between pneumococci occurs during nasopharyngeal colonization, allowing one strain or set of strains to predominate over others. We investigated the contribution of the blp locus, encoding putative bacteriocins and cognate immunity peptides, to intraspecies competition. We sequenced the relevant regions of the blp locus of a type 6A strain able to inhibit the growth of the type 4 strain, TIGR4, in vitro. Using deletional analysis, we confirmed that inhibitory activity is regulated by the function of the response regulator, BlpR, and requires the two putative bacteriocin genes blpM and blpN. Comparison of the TIGR4 BlpM and -N amino acid sequences demonstrated that only five amino acid differences were sufficient to target the heterologous strain. Analysis of a number of clinical isolates suggested that the BlpMN bacteriocins divide into two families. A mutant in the blpMN operon created in the clinically relevant type 19A background was deficient in both bacteriocin activity and immunity. This strain was unable to compete with both its parent strain and a serotype 4 isolate during cocolonization in the mouse nasopharynx, suggesting that the locus is functional in vivo and confirming its role in promoting intraspecies competition.

Expression of a number of eukaryotic genes is regulated by long stretches of tandem repeats located within the 5 untranslated region of the particular gene. In this study, we describe a regulatory system in Haemophilus influenzae with striking similarities to those found in eukaryotes. We show that expression of the HMW1 and HMW2 adhesins varies based on the number of 7-bp tandem repeats in the hmw1A and hmw2A promoters. The repeats lie between two separate transcription initiation sites and exert a repressive effect, such that increases in repeat number result in step-wise decreases in levels of specific mRNA and protein production and vice versa. The range of expression of HMW1 and HMW2 varies between very weak and very strong, with a series of gradations in between. Variation in the number of repeats in the hmw1A and hmw2A promoters occurs in individual colonies passaged in vitro, in an animal model of infection, and during natural infection in humans. This system of regulation is unique in prokaryotes and likely enhances the pathogenicity of the organism by increasing adaptive potential.In recent years, long stretches of tandem oligonucleotide repeats have been found increasingly in the upstream untranslated region of eukaryotic genes. In many cases, the number of repeats influences gene expression, reflecting the fact that transcriptional regulators often bind to the repeat region and regulate transcription (1). For example, the gene encoding human epidermal growth factor receptor is preceded by a series of TCC repeats that are recognized by a transcriptional activator. As these repeats are sequentially deleted, transcriptional activity and protein expression steadily decrease (2). Other examples in which upstream repeats function as transcriptional activating elements include the yeast Adr2p gene, the Drosophila hsp26 and actin 5C genes, the chicken ␣-2(I)collagen gene, and the rat neu gene (3-7). Less commonly, tandem repeats have a negative effect on downstream gene activity (8). Typically, the number of repeats undergoes highfrequency spontaneous variation via slipped-strand mispairing, serving as a source of quantitative genetic variation. Such quantitative variation is believed to confer an evolutionary advantage to a population, facilitating adaptation to novel ecological challenges.Nontypable Haemophilus influenzae is a Gram-negative bacterium that represents a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease (8). The pathogenesis of disease begins with colonization of the upper respiratory mucosa, followed by contiguous spread to the middle ear, the sinuses, or the lungs, usually precipitated by respiratory viral infection or exacerbation of underlying allergic disease (9, 10). Similar to the situation with other bacterial pathogens, adherence to respiratory epithelium is a fundamental step in the process of colonization and is mediated by bacterial surface factors called adhesins. Among diverse strains of nontypabl...

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.

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.

SignificanceThe opportunistic pathogen Streptococcus pneumoniae (pneumococcus) participates in horizontal gene transfer through genetic competence and produces antimicrobial peptides called “bacteriocins.” Here, we show that the competence and bacteriocin-related ABC transporters ComAB and BlpAB share the same substrate pool, resulting in bidirectional crosstalk between competence and bacteriocin regulation. We also clarify the role of each transporter in bacteriocin secretion and show that, based on their transporter content, pneumococcal strains can be separated into a majority opportunist group that uses bacteriocins only to support competence and a minority aggressor group that uses bacteriocins in broader contexts. Our findings will impact how bacteriocin regulation and production is modeled in the many other bacterial species that use ComAB/BlpAB-type transporters.

All fully sequenced strains of Streptococcus pneumoniae (pneumococcus) contain a version of the blp locus which is responsible for the regulation and secretion of a variable repertoire of pneumococcal bacteriocins called pneumocins and their associated immunity proteins. Pneumocins mediate intra-and interspecies competition in vitro and have been shown to provide a competitive advantage in vivo. Pneumocin production is stimulated by extracellular accumulation of the peptide pheromone, BlpC. Both BlpC and the functional pneumocins are secreted out of the cell via the Blp transporter, BlpAB. The conserved surface-expressed serine protease, HtrA, has been shown to limit activation of the locus and secretion of functional pneumocins. In this work, we demonstrate that htrA mutants stimulate the blp locus at lower cell density and to a greater extent than strains expressing wild-type HtrA. This effect is not due to direct proteolytic degradation of secreted pheromone by the protease, but instead is a result of HtrA-mediated disruption of peptide processing and secretion. Because pneumocins are secreted through the same transporter as the pheromone, this finding explains why pheromone supplementation cannot completely restore pneumocin inhibition to strains expressing high levels of HtrA despite restoration of blp transcriptional activity. HtrA restricts pneumocin production to high cell density by limiting the rate of accumulation of BlpC in the environment. Importantly, HtrA does not interfere with the ability of a strain to sense environmental pheromones, which is necessary for the induction of protective immunity in the face of pneumocin-secreting competitors.

The blp locus of a type 6A strain of Streptococcus pneumoniae encodes a two-peptide bacteriocin, pneumocin MN, which mediates intraspecies competition during mouse nasopharyngeal colonization. This locus is regulated by a quorum-sensing mechanism consisting of a dedicated two-component regulatory system and a peptide pheromone. Like most clinical isolates, this type 6A strain can be separated into opaque and transparent colony variants, each playing a different role during pneumococcal infection. In this study, we show that the blp locus is differentially regulated at the posttranscriptional level in pneumococcal opacity variants. Transparent and opaque variants produce equivalent amounts of blpMNPO transcript when stimulated with a synthetic pheromone, but transparent variants have no pneumocin MN-mediated inhibitory activity while opaque variants produce large zones of inhibitory activity. The differential regulation in opacity variants is driven by the two-component regulatory system CiaRH via its regulation of the serine protease HtrA. Transparent mutants deficient in CiaH or HtrA show increased pneumocin MN-mediated inhibition. In addition, these mutants demonstrate alterations in their dose response to a synthetic peptide pheromone, suggesting that HtrA activity impacts pneumocin MN production at the level of signaling. This, in addition to its known effects on competence, suggests that HtrA is a pleiotropic regulator whose protease activity affects several important bacterial pathways. The complex regulation of pneumocins may allow the pneumococcus to reserve the secretion of active peptides for situations where the benefit of their inhibitory activity outweighs the cost of their production.

The blp locus of Streptococcus pneumoniae secretes and regulates bacteriocins, which mediate both intra-and interspecific competition in the human nasopharynx. There are four major alleles of the gene blpH, which encodes the receptor responsible for activating the blp locus when bound to one of four distinct peptide pheromones (BlpC). The allelic variation of blpH is presumably explained by a need to restrict cross talk between competing strains. The BlpH protein sequences have polymorphisms distributed throughout the sequence, making identification of the peptide binding site difficult to predict. To identify the pheromone binding sites that dictate pheromone specificity, we have characterized the four major variants and two naturally occurring chimeric versions of blpH in which recombination events appear to have joined two distinct blpH alleles together. Using these allelic variants, a series of laboratory-generated chimeric blpH alleles, and site-directed mutants of both the receptor and peptide, we have demonstrated that BlpC binding to some BlpH types involves an electrostatic interaction between the oppositely charged residues of BlpC and the first transmembrane domain of BlpH. An additional recognition site was identified in the second extracellular loop. We identified naturally occurring BlpH types that have the capacity to respond to more than one BlpC type; however, this change in specificity results in a commensurate drop in overall sensitivity. These natural recombination events were presumably selected for to balance the need to sense bacteriocin-secreting neighbors with the need to turn on bacteriocin production at a low density. IMPORTANCEBacteria use quorum sensing to optimize gene expression to accommodate for local bacterial density and diffusion rates. To prevent interception of quorum-sensing signals by neighboring strains, the genomes of single species often encode strain-specific signal/receptor pairs. The blp locus in Streptococcus pneumoniae that drives bacteriocin secretion is controlled by quorum sensing that involves the interaction of the signal/receptor pair BlpC/BlpH. We show that the pneumococcal population can be divided into several distinct BlpC/BlpH pairs; however, there are examples of naturally occurring chimeric receptors that can bind to more than one BlpC type. The trade-off for this broadened specificity is a loss of overall receptor sensitivity. This suggests that under certain conditions, the advantage of signal interception can trump the requirements for self-induction. Streptococcus pneumoniae (pneumococcus) is a common pediatric pathogen that colonizes the nasopharynx of a majority of children by 1 year of age (1, 2). During this first year of life, children may be colonized at any point with as many as four different pneumococcal serotypes (3) and even multiple strains of the same pneumococcal serotype (4). The introduction of the multivalent conjugate pneumococcal vaccine has resulted in decreased colonization with the seven targeted serotypes. These strai...