Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP(Spn) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress ΔgpsB or ΔstkP. These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overproduction of MurZ caused by ΔkhpAB mutations suppressed ΔgpsB or ΔstkP phenotypes to varying extents. ΔgpsB and ΔstkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, ΔgpsB and ΔstkP were not suppressed by ΔclpCP, which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB(Spn), is the only essential requirement for StkP-mediated protein phosphorylation in exponentially growing D39 pneumococcal cells.

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 sigX-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 cross talk 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. IMPORTANCE Natural 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.

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 GAS's 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 tandem mass tag (TMT)-LC-MS/MS analysis on cells in which this system was induced with short hydrophobic peptide (SHP) pheromone or disrupted. Primary findings confirmed that pheromone stimulation in wild-type cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, 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, 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. IMPORTANCE Streptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogenes's 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 Rgg2/3 system, leads to stress responses that are associated with the stringent response. Disruption of stringent response affects the ability of the cell-cell communication system to respond under normally inducing conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to nontraditional sources of stress.

Discovery and Biosynthesis of Streptosactin, a Sactipeptide with an Alternative Topology Encoded by Commensal Bacteria in the Human Microbiome.

Mammalian microbiomes encode thousands of biosynthetic gene clusters (BGCs) and represent a new frontier in natural product research. We recently found an abundance of quorum sensing-regulated BGCs in mammalian microbiome streptococci that code for ribosomally synthesized and post-translationally modified peptides (RiPPs) and contain one or more radical S-adenosylmethionine (RaS) enzymes, a versatile superfamily known to catalyze some of the most unusual reactions in biology. In the current work, we target a widespread group of streptococcal RiPP BGCs and elucidate both the reaction carried out by its encoded RaS enzyme and identify its peptide natural product, which we name streptosactin. Streptosactin is the first sactipeptide identified from Streptococcus spp.; it contains two sequential four amino acid sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays reveal potent but narrow-spectrum activity against the producing strain and its closest relatives that carry the same BGC, suggesting streptosactin may be a long-suspected fratricidal agent of Streptococcus thermophilus. Our results highlight mammalian streptococci as a rich source of unusual enzymatic chemistries and bioactive natural products.

The Opp (AmiACDEF) Oligopeptide Transporter Mediates Resistance of Serotype 2 Streptococcus pneumoniae D39 to Killing by Chemokine CXCL10 and Other Antimicrobial Peptides.

Antimicrobial peptides (AMPs), including chemokines, are produced during infections to kill pathogenic bacteria. To fill in gaps in knowledge about the sensitivities of Streptococcus pneumoniae and related Streptococcus species to chemokines and AMPs, we performed a systematic, quantitative study of inhibition by chemokine CXCL10 and the AMPs LL-37 and nisin. In a standard Tris-glucose buffer (TGS), all strains assayed lacked metabolic activity, as determined by resazurin (alamarBlue) reduction, and were extremely sensitive to CXCL10 and AMPs (50% inhibitory concentration [IC50], ∼0.04 µM). In TGS, changes in sensitivities caused by mutations were undetectable. In contrast, strains that retained reductive metabolic activity in a different assay buffer (NPB [10 mM sodium phosphate {pH 7.4}, 1% {vol/vol} brain heart infusion {BHI} broth]) were less sensitive to CXCL10 and AMPs than in TGS. In NPB, mutants known to respond to AMPs, such as Δdlt mutants lacking d-alanylation of teichoic acids, exhibited the expected increased sensitivity. S. pneumoniae serotype 2 strain D39 was much (∼10-fold) less sensitive to CXCL10 killing in NPB than serotype 4 strain TIGR4, and the sensitivity of TIGR4 was unaffected by the absence of capsule. Candidate screening of strain D39 revealed that mutants lacking Opp (ΔamiACDEF) oligopeptide permease were significantly more resistant to CXCL10 than the wild-type strain. This increased resistance could indicate that Opp is a target for CXCL10 binding or that it transports CXCL10 into cells. Finally, ΔftsX or ΔftsE mutants of Bacillus subtilis or amino acid changes that interfere with FtsX function in S. pneumoniae did not impart resistance to CXCL10, in contrast to previous results for Bacillus anthracis, indicating that FtsX is not a general target for CXCL10 binding.IMPORTANCES. pneumoniae (pneumococcus) is a human commensal bacterium and major opportunistic respiratory pathogen that causes serious invasive diseases, killing millions of people worldwide annually. Because of its increasing antibiotic resistance, S. pneumoniae is now listed as a "superbug" for which new antibiotics are urgently needed. This report fills in knowledge gaps and resolves inconsistencies in the scientific literature about the sensitivity of S. pneumoniae and related Streptococcus pathogens to chemokines and AMPs. It also reveals a new mechanism by which S. pneumoniae can acquire resistance to chemokine CXCL10. This mechanism involves the Opp (AmiACDEF) oligopeptide transporter, which plays additional pleiotropic roles in pneumococcal physiology, quorum sensing, and virulence. Taking the results together, this work provides new information about the way chemokines kill pneumococcal cells.

Suppression and synthetic-lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39.

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39.

In ellipsoid-shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The 'synthetic viable' genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic-site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo-lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.

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 ( c ompetence s timulating p eptide, encoded by comC ) and XIP ( sig X -inducing p eptide, 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. IMPORTANCE: Natural 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.

Chromosomal Duplications of MurZ (MurA2) or MurA (MurA1), Amino Acid Substitutions in MurZ (MurA2), and Absence of KhpAB Obviate the Requirement for Protein Phosphorylation in Streptococcus pneumoniae D39.

GpsB links peptidoglycan synthases to other proteins that determine the shape of the respiratory pathogen Streptococcus pneumoniae (pneumococcus; Spn ) and other low-GC Gram-positive bacteria. GpsB is also required for phosphorylation of proteins by the essential StkP( Spn ) Ser/Thr protein kinase. Here we report three classes of frequently arising chromosomal duplications (≈21-176 genes) containing murZ (MurZ-family homolog of MurA) or murA that suppress Δ gpsB or Δ stkP . These duplications arose from three different repeated sequences and demonstrate the facility of pneumococcus to modulate gene dosage of numerous genes. Overproduction of MurZ or MurA alone or overexpression of MurZ caused by Δ khpAB mutations suppressed Δ gpsB or Δ stkP phenotypes to varying extents. Δ gpsB and Δ stkP were also suppressed by MurZ amino-acid changes distant from the active site, including one in commonly studied laboratory strains, and by truncation or deletion of the homolog of IreB(ReoM). Unlike in other Gram-positive bacteria, MurZ is predominant to MurA in pneumococcal cells. However, Δ gpsB and Δ stkP were not suppressed by Δ clpCP , which did not alter MurZ or MurA amounts. These results support a model in which regulation of MurZ and MurA activity, likely by IreB( Spn ), is the only essential requirement for protein phosphorylation in exponentially growing D39 pneumococcal cells.

Quorum Sensing in Streptococcus mutans Regulates Production of Tryglysin, a Novel RaS-RiPP Antimicrobial Compound.

The genus Streptococcus encompasses a large bacterial taxon that commonly colonizes mucosal surfaces of vertebrates and is capable of disease etiologies originating from diverse body sites, including the respiratory, digestive, and reproductive tracts. Identifying new modes of treating infections is of increasing importance, as antibiotic resistance has escalated. Streptococcus mutans is an important opportunistic pathogen that is an agent of dental caries and is capable of systemic diseases such as endocarditis. As such, understanding how it regulates virulence and competes in the oral niche is a priority in developing strategies to defend from these pathogens. We determined that S. mutans UA159 possesses a bona fide short hydrophobic peptide (SHP)/Rgg quorum-sensing system that regulates a specialized biosynthetic operon featuring a radical-SAM (S-adenosyl-l-methionine) (RaS) enzyme and produces a ribosomally synthesized and posttranslationally modified peptide (RiPP). The pairing of SHP/Rgg regulatory systems with RaS biosynthetic operons is conserved across streptococci, and a locus similar to that in S. mutans is found in Streptococcus ferus, an oral streptococcus isolated from wild rats. We identified the RaS-RiPP product from this operon and solved its structure using a combination of analytical methods; we term these RiPPs tryglysin A and B for the unusual Trp-Gly-Lys linkage. We report that tryglysins specifically inhibit the growth of other streptococci, but not other Gram-positive bacteria such as Enterococcus faecalis or Lactococcus lactis We predict that tryglysin is produced by S. mutans in its oral niche, thus inhibiting the growth of competing species, including several medically relevant streptococci.IMPORTANCE Bacteria interact and compete with a large community of organisms in their natural environment. Streptococcus mutans is one such organism, and it is an important member of the oral microbiota. We found that S. mutans uses a quorum-sensing system to regulate production of a novel posttranslationally modified peptide capable of inhibiting growth of several streptococcal species. We find inhibitory properties of a similar peptide produced by S. ferus and predict that these peptides play a role in interspecies competition in the oral niche.

Structure of the Large Extracellular Loop of FtsX and Its Interaction with the Essential Peptidoglycan Hydrolase PcsB in Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended ß-hairpin and a lower α-helical lobe, each extending from a mixed α-ß core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscBCC). Characterization of S. pneumoniae strain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniaeIMPORTANCE FtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as "mechanotransmitters," proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogen S.pneumoniae We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.