RESUMO
Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Compostos Férricos , Interações entre Hospedeiro e Microrganismos , Ferro , Organelas , Animais , Camundongos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/imunologia , Clostridioides difficile/metabolismo , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Ferro/metabolismo , Organelas/metabolismo , Homeostase , Compostos Férricos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Modelos Animais de Doenças , Complexo Antígeno L1 Leucocitário/metabolismo , Viabilidade Microbiana , Inflamação/metabolismo , Inflamação/microbiologia , Intestinos/metabolismo , Intestinos/microbiologiaRESUMO
Bacillus anthracis is the causative agent of anthrax. This Gram-positive bacterium poses a substantial risk to human health due to high mortality rates and the potential for malicious use as a bioterror weapon. To survive within the vertebrate host, B. anthracis relies on two-component system (TCS) signaling to sense host-induced stresses and respond to alterations in the environment through changes in target gene expression. HitRS and HssRS are cross-regulating TCSs in B. anthracis that respond to cell envelope disruptions and high heme levels, respectively. In this study, an unbiased and targeted genetic selection was designed to identify gene products that are involved in HitRS and HssRS signaling. This selection led to the identification of inactivating mutations within dnaJ and clpX that disrupt HitRS- and HssRS-dependent gene expression. DnaJ and ClpX are the substrate-binding subunits of the DnaJK protein chaperone and ClpXP protease, respectively. DnaJ regulates the levels of HitR and HitS to facilitate signal transduction, while ClpX specifically regulates HitS levels. Together, these results reveal that the protein homeostasis regulators, DnaJ and ClpX, function to maintain B. anthracis signal transduction activities through TCS regulation.
Assuntos
Antraz/microbiologia , Bacillus anthracis/fisiologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Transdução de Sinais , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Transporte Proteico , Seleção GenéticaRESUMO
Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.
Assuntos
Bacillus anthracis/fisiologia , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Transdução de Sinais/fisiologia , Seleção Genética/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Transition metals are essential for life but are toxic when in excess. Metal ion intoxication may result from the mismetallation of essential metal-dependent enzymes with a noncognate metal. To begin to identify enzymes and processes that are susceptible to mismetallation, we have selected for strains with increased resistance to Mn(II) and Co(II). In Bacillus subtilis, cells lacking the MntR metalloregulator are exquisitely sensitive to Mn(II) but can easily become resistant by acquiring mutations affecting the MntH Mn(II) importer. Using transposon mutagenesis, and starting with an mntR mntH strain, we recovered mariner insertions that inactivated the mpfA gene encoding a putative Mg(II) efflux system. Loss of MpfA leads to elevated intracellular Mg(II), increased sensitivity to high Mg(II), and reduced Mn(II) sensitivity. Consistently, we also recovered an insertion disrupting the mgtE riboswitch, which normally restricts expression of the major Mg(II) importer. These results suggest that Mn(II) intoxication results from disruption of a Mg(II)-dependent enzyme or process. Mutations that inactivate MpfA were also recovered in a selection for Co(II) resistance beginning with sensitized strains lacking the major Co(II) efflux pump, CzcD. Since both Mn(II) and Co(II) may mismetallate iron-dependent enzymes, we repeated the selections under conditions of iron depletion imposed by expression of the Listeria monocytogenes FrvA iron exporter. Under conditions of iron depletion, a wider variety of suppressor mutations were recovered, but they still point to a central role for Mg(II) in maintaining metal ion homeostasis.IMPORTANCE Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA, encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals.
Assuntos
Bacillus subtilis/metabolismo , Cobalto/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Adaptação Biológica , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Modelos Biológicos , ÓperonRESUMO
Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe2+ efflux transporter from Listeria monocytogenes, as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron (efeUOB), ferric citrate (fecCDEF), and petrobactin (fpbNOPQ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin (dhbACEBF) and turns on bacillibactin (feuABC) and hydroxamate siderophore (fhuBCGD) uptake systems to scavenge iron from the environment and flavodoxins (ykuNOP) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response (fsrA, fbpAB, and fbpC) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Proteínas Repressoras/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Benzamidas/metabolismo , Proteínas de Transporte/metabolismo , Compostos Férricos/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Homeostase/genética , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Oligopeptídeos/biossíntese , Oligopeptídeos/genética , Óperon , Regulon , Proteínas Repressoras/metabolismo , Sideróforos/biossíntese , Sideróforos/genéticaRESUMO
Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection.
Assuntos
Adenosina Trifosfatases/metabolismo , Compostos Ferrosos/metabolismo , Listeria monocytogenes/metabolismo , Fatores de Virulência/metabolismo , Adenosina Trifosfatases/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos Ferrosos/toxicidade , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/enzimologia , Listeria monocytogenes/genética , Mutação , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas Repressoras/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Quinazolinonas/farmacologia , Antibacterianos/farmacocinética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Disponibilidade Biológica , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas de Ligação às Penicilinas , Conformação Proteica , Quinazolinonas/farmacocinética , Staphylococcus/efeitos dos fármacosRESUMO
Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-ß-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Bactérias Gram-Positivas/efeitos dos fármacos , Oxidiazóis/farmacologia , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , beta-Lactamas/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Disponibilidade Biológica , Parede Celular/efeitos dos fármacos , Simulação por Computador , Bactérias Gram-Positivas/citologia , Bactérias Gram-Positivas/metabolismo , Staphylococcus aureus Resistente à Meticilina/citologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/farmacocinética , Proteínas de Ligação às Penicilinas/química , Conformação Proteica , beta-Lactamas/química , beta-Lactamas/farmacocinéticaRESUMO
Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies.
RESUMO
The reactions of all seven Escherichia coli lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall.
Assuntos
Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glicosídeo Hidrolases/metabolismo , Parede Celular/química , Parede Celular/enzimologia , Escherichia coli/citologia , Conformação Molecular , Sáculo e Utrículo/química , Sáculo e Utrículo/metabolismoRESUMO
We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [(59) Fe]-Hn transport, and purified Lmo2185 bound [(59) Fe]-Hn (K(D) = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [(59) Fe]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (K(D) = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (K(D) = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [(59) Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K(M) of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (V(max) = 23 pmol 10(9) cells(-1) min(-1)) to those of ferric siderophore transporters. In the ΔhupDGC strain uptake occurred at a threefold lower rate (V(max) = 7 pmol 10(9) cells(-1) min(-1)). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD(50) of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Heme/metabolismo , Hemoglobinas/metabolismo , Listeria monocytogenes/metabolismo , Aminoaciltransferases/genética , Animais , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Feminino , Humanos , Listeria monocytogenes/enzimologia , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Camundongos , Óperon , VirulênciaRESUMO
HitRS is a two-component system that responds to cell envelope damage in the human pathogen Bacillus anthracis. Here we identify an RNA-binding protein, KrrA, that regulates HitRS function by modulating the stability of the hitRS mRNA. In addition to hitRS, KrrA binds to over 70 RNAs and, directly or indirectly, affects the expression of over 150 genes involved in multiple processes, including genetic competence, sporulation, RNA turnover, DNA repair, transport, and cellular metabolism. KrrA does not exhibit detectable nuclease activity in vitro, and thus the mechanism by which it modulates mRNA stability remains unclear.
Assuntos
Bacillus anthracis , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.
Assuntos
Acinetobacter baumannii , Dessecação , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Pentético/metabolismo , VirulênciaRESUMO
When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB ( approximately 35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of approximately 5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33-35 kcal/mol. The accumulation of FeEnt in the periplasm required the binding protein and inner membrane permease components of its overall transport system; postuptake binding assays on strains devoid of FepB, FepD, or FepG did not show uptake of FeEnt through the OM. However, fluorescence labeling data implied that FepA was active in the DeltafepB strain, suggesting that FeEnt entered the periplasm but then leaked out. Further experiments confirmed this futile cycle; cells without FepB transported FeEnt across the OM, but it immediately escaped through TolC.
Assuntos
Enterobactina/química , Escherichia coli/metabolismo , Ferro/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Imunoprecipitação , Cinética , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Modelos Químicos , Periplasma/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismoRESUMO
Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis: import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg2+ and K+ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K+), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K+ import there is a large reduction in intracellular magnesium (Mg2+) mediated by specific efflux pumps. Further, it is the reimport of Mg2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K+ and Mg2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.
Assuntos
Desidratação , Magnésio , Humanos , Pressão Osmótica , Homeostase , AMP Cíclico/metabolismo , Potássio/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
The ferric uptake regulator (Fur) is the global iron biosensor in many bacteria. Fur functions as an iron-dependent transcriptional repressor for most of its regulated genes. There are a few examples where holo-Fur activates transcription, either directly or indirectly. Recent studies suggest that apo-Fur might also act as a positive regulator and that, besides iron metabolism, the Fur regulon might encompass other biological processes such as DNA synthesis, energy metabolism, and biofilm formation. Here, we obtained a genomic view of the Fur regulatory network in Bacillus subtilis using chromatin immunoprecipitation sequencing (ChIP-seq). Besides the known Fur target sites, 70 putative DNA binding sites were identified, and the vast majority had higher occupancy under iron-sufficient conditions. Among the new sites detected, a Fur binding site in the promoter region of the catDE operon is of particular interest. This operon, encoding catechol 2,3-dioxygenase, is critical for catechol degradation and is under negative regulation of CatR and YodB. These three repressors (Fur, CatR, and YodB) function cooperatively to regulate the transcription of catDE, with Fur functioning as a sensor of iron limitation and CatR as the major sensor of catechol stress. Genetic analysis suggests that CatDE is involved in metabolism of the catecholate siderophore bacillibactin, particularly when bacillibactin is constitutively produced and accumulates intracellularly, potentially generating endogenous toxic catechol derivatives. This study documents a role for catechol degradation in bacillibactin metabolism and provides evidence that catechol 2,3-dioxygenase can detoxify endogenously produced catechol substrates in addition to its more widely studied role in biodegradation of environmental aromatic compounds and pollutants.IMPORTANCE Many bacteria synthesize high-affinity iron chelators (siderophores). Siderophore-mediated iron acquisition is an efficient and widely utilized strategy for bacteria to meet their cellular iron requirements. One prominent class of siderophores uses catecholate groups to chelate iron. B. subtilis bacillibactin, structurally similar to enterobactin (made by enteric bacteria), is a triscatecholate siderophore that is hydrolyzed to monomeric units after import to release iron. However, the ultimate fates of these catechol compounds and their potential toxicities have not been defined previously. We performed genome-wide identification of Fur binding sites in vivo and uncovered a connection between catechol degradation and bacillibactin metabolism in B. subtilis Besides its role in the detoxification of environmental catechols, the catechol 2,3-dioxygenase encoded by catDE also protects cells from intoxication by endogenous bacillibactin-derived catechol metabolites under iron-limited conditions. These findings shed light on the degradation pathway and precursor recycling of the catecholate siderophores.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Catecóis/metabolismo , Regulação Bacteriana da Expressão Gênica , Oligopeptídeos/metabolismo , Proteínas Repressoras/metabolismo , Sítios de Ligação , Biotransformação , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Regiões Promotoras Genéticas , Ligação Proteica , Análise de Sequência de DNARESUMO
Plant growth promoting rhizobacteria (PGPR) provide an effective and environmentally sustainable method to protect crops against pathogens. The spore-forming Bacilli are attractive PGPR due to their ease of storage and application. Here, we characterized two rhizosphere-associated Bacillus velezensis isolates (Y6 and F7) that possess strong antagonistic activity against Ralstonia solanacearum and Fusarium oxysporum under both laboratory and greenhouse conditions. We identified three lipopeptide (LP) compounds (surfactin, iturin and fengycin) as responsible for the antimicrobial activity of these two strains. We further dissected the contribution of LPs to various biological processes important for rhizosphere colonization. Although either iturin or fengycin is sufficient for antibacterial activity, cell motility and biofilm formation, only iturin plays a primary role in defense against the fungal pathogen F. oxysporum. Additionally, we found that LP production is significantly stimulated during interaction with R. solanacearum. These results demonstrate the different roles of LPs in the biology of B. velezensis and highlight the potential of these two isolates as biocontrol agents against phytopathogens.
Assuntos
Bacillus/patogenicidade , Fusarium/efeitos dos fármacos , Lipopeptídeos/toxicidade , Ralstonia solanacearum/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Bacillus/química , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , RizosferaRESUMO
Bacteria require iron for growth, with only a few reported exceptions. In many environments, iron is a limiting nutrient for growth and high affinity uptake systems play a central role in iron homeostasis. However, iron can also be detrimental to cells when it is present in excess, particularly under aerobic conditions where its participation in Fenton chemistry generates highly reactive hydroxyl radicals. Recent results have revealed a critical role for iron efflux transporters in protecting bacteria from iron intoxication. Systems that efflux iron are widely distributed amongst bacteria and fall into several categories: P1B-type ATPases, cation diffusion facilitator (CDF) proteins, major facilitator superfamily (MFS) proteins, and membrane bound ferritin-like proteins. Here, we review the emerging role of iron export in both iron homeostasis and as part of the adaptive response to oxidative stress.
Assuntos
Bactérias/metabolismo , Ferro/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , DifusãoRESUMO
The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.
Assuntos
Antibacterianos/farmacologia , Oxidiazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-AtividadeRESUMO
A complication of diabetes is the inability of wounds to heal in diabetic patients. Diabetic wounds are refractory to healing due to the involvement of activated matrix metalloproteinases (MMPs), which remodel the tissue resulting in apoptosis. There are no readily available methods that identify active unregulated MMPs. With the use of a novel inhibitor-tethered resin that binds exclusively to the active forms of MMPs, coupled with proteomics, we quantified MMP-8 and MMP-9 in a mouse model of diabetic wounds. Topical treatment with a selective MMP-9 inhibitor led to acceleration of wound healing, re-epithelialization, and significantly attenuated apoptosis. In contrast, selective pharmacological inhibition of MMP-8 delayed wound healing, decreased re-epithelialization, and exhibited high apoptosis. The MMP-9 activity makes the wounds refractory to healing, whereas that of MMP-8 is beneficial. The treatment of diabetic wounds with a selective MMP-9 inhibitor holds great promise in providing heretofore-unavailable opportunities for intervention of this disease.