RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus

Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H–13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

High-resolution two-dimensional (2D) 1H–13C heteronuclear correlation spectra are recorded for selective observation of interfacial 3–5.5 Å contacts of the uniformly 13C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI–HETCOR) method. For improving 1H spectral resolution, a windowed phase-modulated Lee–Goldburg (wPMLG) decoupling scheme is applied during the t1 evolution period. Our approach allows for identification of chromophore–protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1–514, Cph1Δ2) containing uniformly 13C- and 15N-labeled PCB chromophore (u-[13C,15N]-PCB-Cph1Δ2) at 17.6 T

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation

In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments

Pneumococcal capsule expression is controlled through a conserved, distal cis-regulatory element during infection

Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial pneumonia in the US and worldwide. Studies have shown that the differing chemical make-up between serotypes of its most important virulence factor, the capsule, can dictate disease severity. Here we demonstrate that control of capsule synthesis is also critical for infection and facilitated by two broadly conserved transcription factors, SpxR and CpsR, through a distal cis-regulatory element we name the 37-CE. Strikingly, changing only three nucleotides within this sequence is sufficient to render pneumococcus avirulent. Using in vivo and in vitro approaches, we present a model where SpxR interacts as a unique trimeric quaternary structure with the 37-CE to enable capsule repression in the airways. Considering its dramatic effect on infection, variation of the 37-CE between serotypes suggests this molecular switch could be a critical contributing factor to this pathogen's serotype-specific disease outcomes

The Phosphoryl Transfer Domain of UhpB Interacts with the Response Regulator UhpA

Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription. UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT. Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione S-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of uhpT expression in a uhp(+) strain. This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation. The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation. Point mutations which allow GST-Bc to activate uhpT expression or which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain. The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR. The GST-Bc protein accelerated dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313. Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred. Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA