Biology subjects

Federle, M. J.

Publications and source records attributed to Federle, M. J..

14 recordsLinked to original sources

bioRxiv2026

Discovery and characterization of a pigment produced by Streptococcus pyogenes

Streptococcus pyogenes is a major human-restricted pathogen capable of both localized and systemic diseases, as well as causing post-infectious acute and chronic rheumatological conditions. Despite its well-documented clinical importance and over a century of extensive research, gaps persist in understanding this pathogen. We report the discovery of a novel pigment produced by S. pyogenes when cultured in a replete, chemically defined medium. Color development accumulates during growth, requires exposure to oxygen, and remains associated with the bacterial cell. Though only 20% of a small strain collection produced the pigment (8 of 40), positive cultures were overrepresented by M1 and M89 serotypes. Given that pigments are critical virulence and fitness determinants in pathogens like Staphylococcus aureus and Streptococcus agalactiae, here we describe initial attempts to characterize S. pyogenes pigment biosynthesis, regulation, and potential benefits to the organism. 20,405 transposon mutants were screened for pigment loss in liquid culture, and we identified 94 independent hits enriched in pathways associated with isoprenoid biosynthesis, purine biosynthesis, guanosine transport, and mixed acid fermentation. Although color development requires oxygen, the extracted pigment did not provide antioxidative activity as compared to non-pigmented extracts. Pigment production was inhibited when the Rgg2/Rgg3 quorum-sensing system was active, though by unknown means. Treatment of RAW-Blue macrophages with the extracted pigment significantly reduced NF{kappa}B activation, suggesting a potential anti-inflammatory effect. Overall, this study describes a previously uncharacterized pigment produced by Streptococcus pyogenes and provides insights into its oxygen-dependent production and associated metabolic pathways. SIGNIFICANCEStreptococcus pyogenes is a ubiquitous pathogen responsible for [~]1.8 million severe infections and 500,000 deaths annually. This organism has not been recognized as a pigment-producing bacterium, but here we identify and characterize the production of a previously unreported colorful compound generated when cultured in a chemically defined medium. This finding reveals an underexplored aspect of S. pyogenes biology and raises the question of its contribution to pathogenesis. Given the established roles of microbial pigments in virulence and immune modulation in other pathogens, this work provides a foundation for future investigations into its functional significance and potential as a target for therapeutic or vaccine development.

microbiology↗

bioRxiv2026

A teichoic acid-like wall modification associated with immune suppression is socially regulated in Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N-acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCEStreptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

microbiology↗

bioRxiv2025

Unraveling Streptococcus pyogenes Carriage: Genomic and Transcriptomic Insights from Acute and Post-Treatment Phases

The carrier state of Streptococcus pyogenes (Group A Streptococcus, GAS), is defined by the presence of the organism without symptoms or an immune response. Carriage poses challenges for clinical management and contributes to transmission in the community. Prior studies suggest genetic and phenotypic differences between isolates from symptomatic (acute) infections and those from carriers, yet comprehensive genomic and transcriptomic analyses of naturally acquired human GAS carriage have been lacking. In this study, we collected longitudinal samples from individuals before and after antibiotic treatment for acute pharyngitis, performing whole genome sequencing and RNA-seq on selected isolates. This is the first report of transcriptional profiling of GAS directly from human derived oropharyngeal swabs. Genomic analysis revealed no consistent carrier-specific genotypes, and in vitro assays showed no major differences in biofilm formation or antibiotic susceptibility between carrier and acute isolates. Transcriptional profiling from oropharyngeal swabs identified distinct gene expression patterns when comparing the acute infection and post-treatment carriage phases, although early acute expression profiles did not predict treatment failure. These findings suggest that GAS carriage is likely influenced by conserved bacterial traits as well as host factors, advancing our understanding of GAS persistence and transmission. SummaryLongitudinal samples were collected from individuals before and after treatment for acute Group A Streptococcal pharyngitis. Whole genome sequencing and RNA-seq was performed on selected isolates. This is the first report of transcriptional profiling of GAS directly from human oropharyngeal swabs. Distinct gene expression patterns were observed when comparing the acute infection and post-treatment carriage.

microbiology↗

bioRxiv2025

Impact of the RaS-RiPP tryglysin and culturing conditions on ex-vivo oral microbiomes.

Natural products are ubiquitously produced by many species that we encounter during our daily lives. One genus, Streptococcus, can produce a wide array of quorum sensing linked natural products known as RaS-RiPPs (ribosomally synthesized and post-translationally modified peptides). Their production is triggered by the induction of an Rgg-SHP quorum sensing system, which senses the presence of SHPs (short hydrophobic peptides) and induces the gene expression of these operons. Previous work has found that streptococcal RaS-RiPPs modulate the growth of different streptococci and might play a role in antibiotic tolerance. This is of particular importance to the oral microbiome, where streptococci are a predominant genus. This study examines the impact of the RaS-RiPP Tryglysin A on ex-vivo oral systems and provides the groundwork for determining how producer Streptococci might impact the composition and function of oral microbiome communities. We additionally explore important factors to consider when culturing ex-vivo oral systems.

microbiology↗

bioRxiv2025

Suppressed macrophage responses to quorum-sensing-active Streptococcus pyogenes occurs at the level of the nucleus

Streptococcus pyogenes, or Group A Streptococus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation in order to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NF{kappa}B activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remains unknown. In this study, we used transcriptomic and phosphoproteomic approaches to address these unanswered questions. We found QS-ON GAS broadly suppressed most inflammatory transcriptional pathways including those of NF{kappa}B, type I and type II interferon responses, and intracellular stress responses. Yet, we found no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NF{kappa}B and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. To determine if epigenetic regulation was involved in QS-mediated immunomodulation, DNA methylation was measured and studies were performed inhibiting various histone and chromatin modifiers. These studies also showed no dijerence between QS-ON compared with QS-OFF infected macrophages. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ unusual methods of restricting inflammation. Uncovering this mechanism will ojer invaluable insight into GAS, itself, as well as understudied immunological pathways. ImportanceStreptococcus pyogenes is a ubiquitous pathogen that causes over 600 million infections every year and 500 thousand to 1 million fatalities. While in developed countries it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections such as necrotizing fasciitis, septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses can be critical in understanding its pathogenicity, epidemiology, and identification of novel treatment avenues in this era of multi-drug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that bacteria can use or inhibit for its survival.

immunology↗

bioRxiv2023

Rgg2/Rgg3 quorum sensing is a determinant of Streptococcus pyogenes-host interactions in a murine intact skin infection model

Streptococcus pyogenes is an obligate human pathobiont associated with many disease states. Here, we present a novel model of S. pyogenes infection using intact murine epithelium. From this model, we were able to perform RNA sequencing to evaluate the genetic changes undertaken by both the bacterium and host at 5- and 24-hours post infection. Analysis of these genomic data demonstrate that S. pyogenes undergoes significant genetic adaptation to successfully infect the murine epithelium, including changes to metabolism and activation of the Rgg2/Rgg3 quorum sensing (QS) system. Subsequent experiments demonstrate that an intact Rgg2/Rgg3 QS cascade is necessary to establish a stable superficial skin infection. Furthermore, activation of this pathway results in increased murine morbidity and increased bacterial burden on the skin. This phenotype is associated with gross changes to the murine skin, as well as histopathological evidence of inflammation. Taken together, these experiments offer a novel method to investigate S. pyogenes-epithelial interactions and demonstrate that a well-studied QS pathway is critical to a persistent infection. ImportanceStreptococcus pyogenes remains a pathogen of global importance, with significant total disease burden worldwide. Much of this burden is due to skin infection or sequalae thereof, yet little is currently known about the initial interactions between the organism and host skin. Here we present a new mouse model of skin infection. From this model, we were able to study gene expression by both the bacteria and the host during early infection time points. Both genetic and phenotypic data derived from these results demonstrate that a well-conserved S. pyogenes communication network, the Rgg2/Rgg3 quorum sensing pathway, contributes to establishing and maintaining a durable skin infection. We propose that by better understanding the genetic pathways needed to colonize and adapt to new niches, new approaches to preventing and treating S. pyogenes may be possible.

microbiology↗

bioRxiv2023

The ComRS-SigX pathway regulates natural transformation in Streptococcus ferus

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing competence-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that crosstalk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCENatural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

microbiology↗

bioRxiv2022

The proteomic and transcriptomic landscapes altered by Rgg2/3 activity in Streptococcus pyogenes

Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GASs ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and TMT-LC-MS/MS analysis on cells in which this system was induced or disrupted. Primary findings confirmed that pheromone stimulation in wildtype cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, supplementing exogenous pheromone to a deletion mutant of rgg3, a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions ({Delta}rgg3+SHP), a connection was identified between Rgg2/3 and the stringent response. Mutation of relA, the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB, impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. IMPORTANCEStreptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogeness gene expression and, in extreme conditions, its physiology. Using transcriptomic and proteomic approaches, the results define the pheromone-dependent regulon of the Rgg2/3 quorum sensing system. In addition, we find that excessive pheromone stimulation, generated by genetic disruption of the system, leads to stress responses that are associated with the stringent response. Disruption of this stress response affects the ability of the cell-cell communication system to respond under certain conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to non-traditional sources of stress.

microbiology↗

bioRxiv2022

Quorum sensing regulation of a major facilitator superfamily transporter affects multiple streptococcal virulence factors

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or Group A strep, one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show that the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. ImportanceThe production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds that expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple secreted virulence factors and accounts for a documented QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between the expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

microbiology↗

bioRxiv2021

The Atypical Antipsychotic Quetiapine Induces Multiple Antibiotic Resistance in Escherichia coli

Atypical antipsychotic (AAP) medication is a critical tool for treating symptoms of psychiatric disorders. While AAPs primarily target dopamine (D2) and serotonin (5HT2A and 5HT1A) receptors, they also exhibit intrinsic antimicrobial activity as an off-target effect. Because AAPs are often prescribed to patients for many years, a potential risk associated with long-term AAP use is the unintended emergence of bacteria with antimicrobial resistance (AMR). Here, we show that exposure to the AAP quetiapine at estimated gut concentrations promotes AMR in Escherichia coli after six weeks. Quetiapine-exposed isolates exhibited an increase in minimal inhibitory concentrations (MICs) for ampicillin, tetracycline, ceftriaxone, and levofloxacin. By whole genome sequencing analysis, we identified mutations in genes that confer AMR, including the repressor for the multiple antibiotic resistance mar operon (marR), and real-time RT-qPCR analysis showed increased levels of marA, acrA, and tolC mRNAs and a reduced level of ompF mRNA in the isolates carrying marR mutations. To determine the contribution of each marR mutation to AMR, we constructed isogenic strains carrying individual mutant marR alleles in the parent background and re-evaluated their resistant phenotypes using MIC and RT-qPCR assays. While marR mutations induced a robust activity of the mar operon, they resulted in only a modest increase in MICs. Interestingly, although these marR mutations did not fully recapitulate the AMR phenotype of the quetiapine-exposed isolates, we show that marR mutations promote growth fitness in the presence of quetiapine. Our findings revealed an important link between the use of AAPs and AMR development in E. coli.

microbiology↗

bioRxiv2021

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

microbiology↗

bioRxiv2020

A Streptococcus quorum sensing system enables suppression of innate immunity

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (Group A Streptococcus, GAS) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF{kappa}B activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence were seen as different between QS activity states, yet TNF, IL-6, and IFN{beta} levels and NF{kappa}B reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. ImportanceStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe post-infectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage pro-inflammatory cytokine responses. Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

microbiology↗

bioRxiv2020

Colonization of the murine oropharynx by Streptococcus pyogenes is governed by the Rgg2/3 quorum sensing system

Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors are known to contribute to persistent colonization of this niche, and many are important in mucosal immunity and vaccine development. In this work, mice were infected intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system--a peptide-based signaling system conserved in sequenced isolates of S. pyogenes. Deletion of the QS systems transcriptional activator ({Delta}rgg2) dramatically diminished the percentage of colonized mice while deletion of the transcriptional repressor ({Delta}rgg3) increased the percentage of colonized mice compared to wild type. Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that QS-dependent colonization is responsive to the intrinsic conditions within the host upper respiratory tract. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative RT-PCR subsequently confirmed QS upregulation within one hour of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the {Delta}rgg2 bacteria suggests that their rapid elimination failed to elicit the previously characterized response to intranasal inoculation of GAS. This work identifies a new transcriptional regulatory system governing the ability of S. pyogenes to colonize the nasopharynx and provides knowledge that could help lead to decolonization therapeutics. Author SummaryStreptococcus pyogenes is responsible for a wide spectrum of diseases ranging from common pharyngitis to infrequent invasive infections like necrotizing fasciitis. The ability of this microorganism to persist in the human oropharynx predisposes colonized individuals to a variety of superficial and invasive diseases which lead to significant morbidities and mortality. Identification of the regulatory systems that augment the bacterias ability to colonize the oropharynx provides potential targets against which molecular therapeutics can be designed. Here we show that the Rgg2/3 quorum sensing system, an interbacterial communication system, governs the ability of S. pyogenes to colonize the murine oropharynx. Disruption of the systems transcriptional activator reduced colonization dramatically, eliminated the transcription of two sets of genes known to be activated by the Rgg2/3 system, and tempered the innate immune response seen when S. pyogenes is intranasally infected into the mouse.

microbiology↗

bioRxiv2020

Structural basis of Rgg protein binding to their regulatory pheromones and target DNA promoters

Rgg family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryo-EM structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures to that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both S. dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of the observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of the SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci. Significance StatementSecreted peptide pheromones regulate critical biological processes in Gram-positive bacteria. In streptococci such as the human pathogen S. pyogenes, oligopeptide pheromones, like the short hydrophobic peptides (SHPs), regulate virulence, antimicrobial resistance, and biofilm formation. SHPs directly regulate the activity of transcription factors called Rgg2 and Rgg3. We present the cryo-EM structure of Rgg3 in complex with SHP3, as well as X-ray crystal structures of Rgg2 bound to target promoter DNA, Rgg3 bound to target promoter DNA, and Rgg3 alone. Based on the cryo-EM, X-ray crystallographic, biochemical, and genetic studies presented here, we provide not only detailed mechanistic insight into the molecular basis of Rgg3-SHP3, Rgg2-DNA, and Rgg3-DNA binding specificity, but also a new model of transcription factor regulatory interplay.

microbiology↗