Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum

Identification and Mechanistic Elucidation of the Rgg2/3 Quorum Sensing Circuit of Streptococcus pyogenes

Bacterial cell to cell communication, known as quorum sensing (QS), exists in a wide range of bacterial species and in many cases the responses elicited by QS signals contribute directly to pathogenesis. Recently, a new quorum sensing system was discovered in multiple streptococcal species in which a peptide pheromone modulates control of target genes through transcriptional regulators belonging to the Rgg-family. The Gram-positive bacterium Streptococcus pyogenes, also known as Group A streptococcus (GAS), is a strictly human commensal that poses large health and economic burdens worldwide deriving from its ability to cause self-limiting acute infections, life-threatening invasive disease, and post-infection sequelae. GAS has no known conserved quorum sensing systems, but has four rgg paralogs encoded in all 13 sequenced genomes. Two of these Rgg proteins (Rgg2 and Rgg3) are located in the genome adjacent to small open reading frames encoding putative peptides (SHP2, SHP3) with similar properties to pheromones of other QS systems. Using a combination of genetic and biochemical approaches, we have identified a novel QS system in GAS which utilizes both Rgg2 and Rgg3 in conjunction with SHP2 and SHP3 to control target gene expression. We demonstrate that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes. The C-terminal eight amino acids of the SHP peptides are identical at seven of eight positions and comprise the active signaling molecules, and both mature peptides function intracellularly to induce system activation through de-repression of Rgg3 and activation of Rgg2. Intriguingly, Rgg2 and Rgg3 share a highly conserved binding site within target promoters, and direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP peptide. Studies presented herein also suggest some degree of Rgg-SHP specificity and demonstrate that both shp gene dosage and identity contribute to system activation. This is a unique regulatory circuit among known peptide-responsive QS systems, and further insight into Rgg function is anticipated to be of large importance to the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species

Regulation and Consequence of Serine Catabolism in Streptococcus pyogenes ▿

The Gram-positive bacterium Streptococcus pyogenes (also called group A Streptococcus [GAS]), is found strictly in humans and is capable of causing a wide variety of infections. Here we demonstrate that serine catabolism in GAS is controlled by the transcriptional regulator Spy49_0126c. We have designated this regulator SerR (for serine catabolism regulator). Microarray and transcriptional reporter data show that SerR acts as a transcriptional repressor of multiple operons, including sloR and sdhBA. Purified recombinant SerR binds to the promoters of both sloR and sdhB, demonstrating that this regulation is direct. Deletion of serR results in a lower culture yield of the mutant than of the wild type when the strains are grown in defined medium unless additional serine is provided, suggesting that regulation of serine metabolism is important for maximizing bacterial growth. Deletion of sloR or sdhB in the ΔserR mutant background restores growth to wild-type levels, suggesting that both operons have roles in serine catabolism. While reports have linked sloR function to streptolysin O expression, transport experiments with radiolabeled l-serine reveal that the sloR operon is required for rapid acquisition of serine, suggesting a novel role for this operon in amino acid metabolism

Recipient-Biased Competition For A Cross-Fed Nutrient Is Required For Coexistence Of Microbial Mutualists

Many mutualistic microbial relationships are based on nutrient cross-feeding. Traditionally, cross-feeding is viewed as being unidirectional, from the producer to the recipient. This is likely true when a producer’s waste, such as a fermentation product, has value only for a recipient. However, in some cases the cross-fed nutrient holds value for both the producer and the recipient. In such cases, there is potential for nutrient reacquisition by producer cells in a population, leading to competition against recipients. Here, we investigated the consequences of interpartner competition for cross-fed nutrients on mutualism dynamics by using an anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli excretes waste organic acids that provide a carbon source for R. palustris. In return, R. palustris cross-feeds E. coli ammonium (NH$_4$$^+$), a compound that both species value. To explore the potential for interpartner competition, we first used a kinetic model to simulate cocultures with varied affinities for NH$_4$$^+$ in each species. The model predicted that interpartner competition for NH$_4$$^+$ could profoundly impact population dynamics. We then experimentally tested the predictions by culturing mutants lacking NH$_4$$^+$ transporters in both NH$_4$$^+$ competition assays and mutualistic cocultures. Both theoretical and experimental results indicated that the recipient must have a competitive advantage in acquiring cross-fed NH$_4$$^+$ to sustain the mutualism. This recipient-biased competitive advantage is predicted to be crucial, particularly when the communally valuable nutrient is generated intracellularly. Thus, the very metabolites that form the basis for mutualistic cross-feeding can also be subject to competition between mutualistic partners

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

Microbial interactions, including mutualistic nutrient exchange (cross-feeding), underpin the flow of energy and materials in all ecosystems. Metabolic exchanges are difficult to assess within natural systems. As such, the impact of exchange levels on ecosystem dynamics and function remains unclear. To assess how cross-feeding levels govern mutualism behavior, we developed a bacterial coculture amenable to both modeling and experimental manipulation. In this coculture, which resembles an anaerobic food web, fermentative Escherichia coli and photoheterotrophic Rhodopseudomonas palustris obligately cross-feed carbon (organic acids) and nitrogen (ammonium). This reciprocal exchange enforced immediate stable coexistence and coupled species growth. Genetic engineering of R. palustris to increase ammonium cross-feeding elicited increased reciprocal organic acid production from E. coli, resulting in culture acidification. Consequently, organic acid function shifted from that of a nutrient to an inhibitor, ultimately biasing species ratios and decreasing carbon transformation efficiency by the community; nonetheless, stable coexistence persisted at a new equilibrium. Thus, disrupting the symmetry of nutrient exchange can amplify alternative roles of an exchanged resource and thereby alter community function. These results have implications for our understanding of mutualistic interactions and the use of microbial consortia as biotechnology

Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism.

Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+ The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a prioriIMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities

Redundant Group A Streptococcus Signaling Peptides Exhibit Unique Activation Potentials

All bacterial quorum sensing (QS) systems are based on the production, secretion, and detection of small signaling molecules. Gram-positive bacteria typically use small peptides as QS effectors, and each QS circuit generally requires the interaction of a single signaling molecule with a single receptor protein. The recently described Rgg2 and Rgg3 (Rgg2/3) regulatory circuit of Streptococcus pyogenes (group A streptococcus [GAS]) is one of only a few QS circuits known to utilize multiple signaling peptides. In this system, two distinct, endogenously produced peptide pheromones (SHP2 and SHP3) both function to activate the QS circuit. The aim of this study was to further define the roles of SHP2 and SHP3 in activation of the Rgg2/3 QS system, specifically with regard to shp gene identity and dosage. Results from our studies using transcriptional reporters and isogenic GAS mutants demonstrate that shp gene dosage does contribute to Rgg2/3 system induction, as decreased gene dosage results in decreased or absent induction. Beyond this, however, data indicate that the shp genes possess distinct potentials for supporting system activation, with shp3 more readily able to support system activation than shp2. Studies using synthetic peptides and shp gene mutants indicate that the disparate activities of endogenous SHPs are due to production, rather than signaling, differences and are conferred by the N-terminal regions rather than the C-terminal signaling regions of the peptides. These data provide evidence that the N-terminal, noneffector sequences of SHP pheromones influence their production efficiencies and thereby the relative activation potentials of endogenous SHPs

Light cues induce protective anticipation of environmental water loss in terrestrial bacteria

The ecological significance of light perception in nonphotosynthetic bacteria remains largely elusive. In terrestrial environments, diurnal oscillations in light are often temporally coupled to other environmental changes, including increased temperature and evaporation. Here, we report that light functions as an anticipatory cue that triggers protective adaptations to tolerate a future rapid loss of environmental water. We demonstrate this photo-anticipatory stress tolerance in leaf-associated Pseudomonas syringae pv. syringae (Pss) and other plant- and soil-associated pseudomonads. We found that light influences the expression of 30% of the Pss genome, indicating that light is a global regulatory signal, and this signaling occurs almost entirely via a bacteriophytochrome photoreceptor that senses red, far-red, and blue wavelengths. Bacteriophytochrome-mediated light control disproportionally up-regulates water-stress adaptation functions and confers enhanced fitness when cells encounter light prior to water limitation. Given the rapid speed at which water can evaporate from leaf surfaces, such anticipatory activation of a protective response enhances fitness beyond that of a reactive stress response alone, with recurring diurnal wet–dry cycles likely further amplifying the fitness advantage over time. These findings demonstrate that nonphotosynthetic bacteria can use light as a cue to mount an adaptive anticipatory response against a physiologically unrelated but ecologically coupled stress.This article is published as Hatfield, Bridget M., Breah LaSarre, Meiling Liu, Haili Dong, Dan Nettleton, and Gwyn A. Beattie. "Light cues induce protective anticipation of environmental water loss in terrestrial bacteria." Proceedings of the National Academy of Sciences 120, no. 38 (2023): e2309632120. Copyright © 2023 the Author(s). Posted with permission.This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivativesLicense 4.0 (CC BY-NC-ND)