Structural and functional characterization of MrpR, the master repressor of the Bacillus subtilis prophage SPß.

Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPß prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPß c2 mutant, we demonstrate that the lytic cycle of SPß c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPß genome. MrpR functions as a master repressor of SPß that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPß c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPß.

HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression.

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.

Impact of CO2/HCO3- Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2 The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains.IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3- levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3- pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

Membrane-Permeable Octanoyloxybenzyl-Masked cNMPs As Novel Tools for Non-Invasive Cell Assays.

Adenine nucleotide (AN) 2nd messengers, such as 3',5'-cyclic adenosine monophosphate (cAMP), are central elements of intracellular signaling, but many details of their underlying processes remain elusive. Like all nucleotides, cyclic nucleotide monophosphates (cNMPs) are net-negatively charged at physiologic pH which limits their applicability in cell-based settings. Thus, many cellular assays rely on sophisticated techniques like microinjection or electroporation. This setup is not feasible for medium- to high-throughput formats, and the mechanic stress that cells are exposed to raises the probability of interfering artefacts or false-positives. Here, we present a short and flexible chemical route yielding membrane-permeable, bio-reversibly masked cNMPs for which we employed the octanoyloxybenzyl (OB) group. We further show hydrolysis studies on chemical stability and enzymatic activation, and present results of real-time assays, where we used cAMP and Ca2+ live cell imaging to demonstrate high permeability and prompt intracellular conversion of some selected masked cNMPs. Based on these results, our novel OB-masked cNMPs constitute valuable precursor-tools for non-invasive studies on intracellular signaling.

Adhesion to laminin-1 and collagen IV induces the formation of Ca2+ microdomains that sensitize mouse T cells for activation.

During an immune response, T cells migrate from blood vessel walls into inflamed tissues by migrating across the endothelium and through extracellular matrix (ECM). Integrins facilitate T cell binding to endothelial cells and ECM proteins. Here, we report that Ca2+ microdomains observed in the absence of T cell receptor (TCR)/CD3 stimulation are initial signaling events triggered by adhesion to ECM proteins that increase the sensitivity of primary murine T cells to activation. Adhesion to the ECM proteins collagen IV and laminin-1 increased the number of Ca2+ microdomains in a manner dependent on the kinase FAK, phospholipase C (PLC), and all three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes and promoted the nuclear translocation of the transcription factor NFAT-1. Mathematical modeling predicted that the formation of adhesion-dependent Ca2+ microdomains required the concerted activity of two to six IP3Rs and ORAI1 channels to achieve the increase in the Ca2+ concentration in the ER-plasma membrane junction that was observed experimentally and that required SOCE. Further, adhesion-dependent Ca2+ microdomains were important for the magnitude of the TCR-induced activation of T cells on collagen IV as assessed by the global Ca2+ response and NFAT-1 nuclear translocation. Thus, adhesion to collagen IV and laminin-1 sensitizes T cells through a mechanism involving the formation of Ca2+ microdomains, and blocking this low-level sensitization decreases T cell activation upon TCR engagement.

A pseudokinase version of the histidine kinase ChrS promotes high heme tolerance of Corynebacterium glutamicum.

Heme is an essential cofactor for almost all living cells by acting as prosthetic group for various proteins or serving as alternative iron source. However, elevated levels are highly toxic for cells. Several corynebacterial species employ two paralogous, heme-responsive two-component systems (TCS), ChrSA and HrrSA, to cope with heme stress and to maintain intracellular heme homeostasis. Significant cross-talk at the level of phosphorylation between these systems was previously demonstrated. In this study, we have performed a laboratory evolution experiment to adapt Corynebacterium glutamicum to increasing heme levels. Isolated strains showed a highly increased tolerance to heme growing at concentrations of up to 100 µM. The strain featuring the highest heme tolerance harbored a frameshift mutation in the catalytical and ATPase-domain (CA-domain) of the chrS gene, converting it into a catalytically-inactive pseudokinase (ChrS_CA-fs). Reintroduction of the respective mutation in the parental C. glutamicum strain confirmed high heme tolerance and showed a drastic upregulation of hrtBA encoding a heme export system, conserved in Firmicutes and Actinobacteria. The strain encoding the ChrS pseudokinase variant showed significantly higher heme tolerance than a strain lacking chrS. Mutational analysis revealed that induction of hrtBA in the evolved strain is solely mediated via the cross-phosphorylation of the response regulator (RR) ChrA by the kinase HrrS and BACTH assays revealed the formation of heterodimers between HrrS and ChrS. Overall, our results emphasize an important role of the ChrS pseudokinase in high heme tolerance of the evolved C. glutamicum and demonstrate the promiscuity in heme-dependent signaling of the paralogous two-component systems facilitating fast adaptation to changing environmental conditions.

The diversity of heme sensor systems - heme-responsive transcriptional regulation mediated by transient heme protein interactions.

Heme is a versatile molecule that is vital for nearly all cellular life by serving as prosthetic group for various enzymes or as nutritional iron source for diverse microbial species. However, elevated levels of heme is toxic to cells. The complexity of this stimulus has shaped the evolution of diverse heme sensor systems, which are involved in heme-dependent transcriptional regulation in eukaryotes and prokaryotes. The functions of these systems are manifold-ranging from the specific control of heme detoxification or uptake systems to the global integration of heme and iron homeostasis. This review focuses on heme sensor systems, regulating heme homeostasis by transient heme protein interaction. We provide an overview of known heme-binding motifs in prokaryotic and eukaryotic transcription factors. Besides the central ligands, the surrounding amino acid environment was shown to play a pivotal role in heme binding. The diversity of heme-regulatory systems, therefore, illustrates that prediction based on pure sequence information is hardly possible and requires careful experimental validation. Comprehensive understanding of heme-regulated processes is not only important for our understanding of cellular physiology, but also provides a basis for the development of novel antibacterial drugs and metabolic engineering strategies.

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction­NAADP to NAADPH­is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.