RESUMEN
Bacterial quinones are lipophilic redox compounds involved in important cellular roles such as electron transport in respiratory and photosynthetic chains. However, a growing body of research has now revealed that quinones have additional functions in bacterial physiology, with significant consequences for colonization and persistence in different ecological niches. The aim of this review is to provide an updated view of the functions of bacterial quinones, with particular emphasis on their importance for bacterial metabolism, gene regulation, and stress resistance. We provide evidence that quinones have also a deep impact on the composition and function of bacterial ecosystems.
Asunto(s)
Electrones , Quinonas , Bacterias/genética , Bacterias/metabolismo , Ecosistema , Transporte de Electrón , Oxidación-Reducción , Quinonas/metabolismoRESUMEN
In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.
Asunto(s)
Proteínas Bacterianas/genética , Productos del Gen rex/genética , NAD/deficiencia , Regulón , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/patogenicidad , Virulencia , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Femenino , Perfilación de la Expresión Génica , Productos del Gen rex/química , Productos del Gen rex/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Conformación Proteica , Infecciones Estreptocócicas/metabolismoRESUMEN
The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.
Asunto(s)
Transporte de Electrón , NADH Deshidrogenasa/metabolismo , Streptococcus agalactiae/enzimología , Streptococcus agalactiae/metabolismo , Factores de Virulencia/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Oxidación-Reducción , Oxígeno/metabolismo , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , Agua/metabolismoRESUMEN
Aerobic respiration metabolism in Group B Streptococcus (GBS) is activated by exogenous heme and menaquinone. This capacity enhances resistance of GBS to acid and oxidative stress and improves its survival. In this work, we discovered that GBS is able to respire in the presence of heme and 1,4-dihydroxy-2-naphthoic acid (DHNA). DHNA is a biosynthetic precursor of demethylmenaquinone (DMK) in many bacterial species. A GBS gene (gbs1789) encodes a homolog of the MenA 1,4-dihydroxy-2-naphthoate prenyltransferase enzyme, involved in the synthesis of demethylmenaquinone. In this study, we showed that gbs1789 is involved in the biosynthesis of long-chain demethylmenaquinones (DMK-10). The Δgbs1789 mutant cannot respire in the presence of heme and DHNA, indicating that endogenously synthesized DMKs are cofactors of the GBS respiratory chain. We also found that isoprenoid side chains from GBS DMKs are produced by the protein encoded by the gbs1783 gene, since this gene can complement an Escherichia coli ispB mutant defective for isoprenoids chain synthesis. In the gut or vaginal microbiote, where interspecies metabolite exchanges occur, this partial DMK biosynthetic pathway can be important for GBS respiration and survival in different niches.
Asunto(s)
Benzoquinonas/metabolismo , Streptococcus agalactiae/metabolismo , Vitamina K 2/metabolismo , Vías Biosintéticas , Hemo/metabolismo , Redes y Vías Metabólicas , Naftoles/metabolismo , Naftoles/farmacología , Streptococcus agalactiae/genética , Vitamina K 2/análogos & derivadosRESUMEN
The interaction between pathogenic microbes and their hosts is determined by survival strategies on both sides. As a result of its redox properties, iron is vital for the growth and proliferation of nearly all organisms, including pathogenic bacteria. In bacteria-vertebrate interactions, competition for this essential metal is critical for the outcome of the infection. The role of iron in the virulence of plant pathogenic bacteria has only been explored in a few pathosystems in the past. However, in the last 5 years, intensive research has provided new insights into the mechanisms of iron homeostasis in phytopathogenic bacteria that are involved in virulence. This review, which includes important plant pathosystems, discusses the recent advances in the understanding of iron transport and homeostasis during plant pathogenesis. By summarizing the recent progress, we wish to provide an updated view clarifying the various roles played by this metal in the virulence of bacterial phytopathogens as a nutritional and regulatory element. The complex intertwining of iron metabolism and oxidative stress during infection is emphasized.
Asunto(s)
Bacterias/metabolismo , Bacterias/patogenicidad , Homeostasis , Hierro/metabolismo , Plantas/microbiología , Estallido Respiratorio , VirulenciaRESUMEN
Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.
Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Enterobacteriaceae/patogenicidad , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Animales , Proteínas Bacterianas/metabolismo , Enterobacteriaceae/química , Enterobacteriaceae/fisiología , Insectos/microbiología , Iones/metabolismo , Pectobacterium/química , Pectobacterium/patogenicidad , Pectobacterium/fisiología , VirulenciaRESUMEN
During infection, the phytopathogenic enterobacterium Dickeya dadantii has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. A tight control of the bacterial intracellular iron content is necessary for full virulence of D. dadantii: previous studies have shown that the ferritin FtnA and the bacterioferrtin Bfr, devoted to iron storage, contribute differentially to the virulence of this species. In this work, we investigated the role of the Dps miniferritin in iron homeostasis in D. dadantii. We constructed a Dps-deficient mutant by reverse genetics. This mutant grew like the wild-type stain under iron starvation and showed no decreased iron content. However, the dps mutant displayed an increased sensitivity to hydrogen peroxide in comparison to the wild-type strain. This hydrogen peroxide susceptibility only occurs when bacteria are in the stationary phase. Unlike the bfr and the ftnA mutants, the dps mutant is not affected in its pathogenicity on host plants. The dps gene expression is induced at the stationary phase of growth. The Sigma S transcriptional factor is necessary for this control. Furthermore, dps expression is positively regulated by the oxidative stress response regulator OxyR during the exponential growth phase, after hydrogen peroxide treatment. These results indicate that the Dps miniferritin from D. dadantii has a minor role in iron homeostasis, but is important in conferring tolerance to hydrogen peroxide and for survival of cells that enter the stationary phase of growth.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Dickeya chrysanthemi/fisiología , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Ferritinas/genética , Hierro/metabolismo , Datos de Secuencia Molecular , Mutación , Estrés OxidativoRESUMEN
Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Enterobacteriaceae/genética , Genoma Bacteriano , Enterobacteriaceae/aislamiento & purificación , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Plantas/microbiología , Análisis de Secuencia de ADNRESUMEN
The intracellular fate of iron acquired by bacteria during siderophore-mediated assimilation is poorly understood. We investigated this question in the pathogenic enterobacterium Erwinia chrysanthemi. This bacterium produces two siderophores, chrysobactin and achromobactin, during plant infection. We analyzed the distribution of iron into cytosolic proteins in bacterial cells supplied with 59Fe-chrysobactin using native gel electrophoresis. A parental strain and mutants deficient in bacterioferritin (bfr), miniferritin (dps), ferritin (ftnA), bacterioferredoxin (bfd), or iron-sulfur cluster assembly machinery (sufABCDSE) were studied. In the parental strain, we observed two rapidly 59Fe-labeled protein signals identified as bacterioferritin and an iron pool associated to the protein chain-elongation process. In the presence of increased 59Fe-chrysobactin concentrations, we detected mini-ferritin-bound iron. Iron incorporation into bacterioferritin was severely reduced in nonpolar sufA, sufB, sufD, sufS, and sufE mutants but not in a sufC background. Iron recycling from bacterioferritin did not occur in bfd and sufC mutants. Iron depletion caused a loss of aconitase activity, whereas ferric chrysobactin supplementation stimulated the production of active aconitase in parental cells and in bfr and bfd mutants. Aconitase activity in sufA, sufB, sufD, sufS, and sufE mutant strains was 10 times lower than that in parental cells. In the sufC mutant, it was twice as low as that in the parental strain. Defects observed in the mutants were not caused by altered ferric chrysobactin transport. Our data demonstrate a functional link between bacterioferritin, bacterioferredoxin, and the Suf protein machinery resulting in optimal bacterial growth and a balanced distribution of iron between essential metalloproteins.
Asunto(s)
Proteínas Bacterianas/química , Grupo Citocromo b/química , Dickeya chrysanthemi/metabolismo , Ferritinas/química , Regulación Bacteriana de la Expresión Génica , Proteínas Hierro-Azufre/química , Hierro/metabolismo , Sideróforos/metabolismo , Proteínas Bacterianas/fisiología , Transporte Biológico , Grupo Citocromo b/fisiología , Dipéptidos/química , Electroforesis en Gel de Poliacrilamida , Ferritinas/fisiología , Hierro/química , Metaloproteínas/química , Modelos Biológicos , Mutación , Oxidación-Reducción , Plásmidos/metabolismoRESUMEN
During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.
Asunto(s)
Proteínas Bacterianas/genética , Grupo Citocromo b/genética , Dickeya chrysanthemi/genética , Ferritinas/genética , Hierro/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Secuencia de Bases , Transporte Biológico , Northern Blotting , Cichorium intybus/microbiología , Cloruros , Grupo Citocromo b/metabolismo , Grupo Citocromo b/fisiología , Dickeya chrysanthemi/metabolismo , Dickeya chrysanthemi/patogenicidad , Compuestos Férricos/metabolismo , Ferritinas/metabolismo , Ferritinas/fisiología , Compuestos Ferrosos/metabolismo , Regulación Bacteriana de la Expresión Génica , Radioisótopos de Hierro/metabolismo , Datos de Secuencia Molecular , Mutación , Estrés Oxidativo , Hojas de la Planta/microbiología , Espectroscopía de Mossbauer , Virulencia/genéticaRESUMEN
The enterobacterial pathogen Erwinia chrysanthemi causes soft rot diseases on a wide range of plants, including the model plant Arabidopsis thaliana. This bacterium proliferates in the host by secreting a set of pectin degrading enzymes responsible for symptom development. In addition, survival of this bacterium in planta requires two high-affinity iron acquisition systems mediated by siderophores and protective systems against oxidative damages, suggesting the implication by both partners of accurate mechanisms controlling their iron homeostasis under conditions of infection. In this review, we address this question and we show that ferritins both from the pathogen and the host are subtly implicated in the control of this interplay.
Asunto(s)
Arabidopsis/inmunología , Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/patogenicidad , Ferritinas/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/citología , Arabidopsis/microbiología , Proteínas Bacterianas/genética , Ferritinas/genética , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Datos de Secuencia MolecularRESUMEN
Full virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae.
Asunto(s)
Citratos/fisiología , Dickeya chrysanthemi/metabolismo , Compuestos de Hierro/metabolismo , Sideróforos/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Citratos/biosíntesis , Clonación Molecular , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/patogenicidad , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Ácidos Cetoglutáricos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Operón , Enfermedades de las Plantas/microbiología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Viola/microbiología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
Two major virulence determinants of the plant-pathogenic enterobacterium Erwinia chrysanthemi strain 3937 are the production of pectate lyase enzymes that degrade plant cell walls and expression of two high-affinity iron uptake systems mediated by two structurally unrelated siderophores, chrysobactin and achromobactin. Low iron availability is a signal that triggers transcription of the genes encoding pectate lyases PelD and PelE as well as that of genes involved in iron transport. This metalloregulation is mediated by the transcriptional repressor Fur. In this study, we analyzed the molecular mechanisms of this control. We purified the Erwinia chrysanthemi Fur protein. Band shift assays showed that Fur specifically binds in vitro to the regulatory regions of the genes encoding the ferrichrysobactin outer membrane receptor Fct and the pectate lyases PelD and PelE. We identified the Fur-binding sites of these promoter regions by performing DNase I footprinting experiments. From these data, we propose that Fur could inhibit the activation of the pelD and pelE genes by the cAMP receptor protein CRP according to an anti-activation mechanism. To identify other possible effectors involved in this control, we screened a bank of insertion mutants for an increase in transcriptional activity of pelD and fct genes in response to iron limitation. We isolated a mutant affected in the kdgK gene encoding the 2-keto-3-deoxygluconate (KDG) kinase, an enzyme involved in pectin catabolism. The growth of this mutant in the presence of pectic compounds led to a constitutive expression of iron transport genes as well as complete derepression of the pectinolysis genes. This effect was caused by intracellular accumulation of KDG. However, the derepression of iron transport genes by KDG does not involve the KdgR regulator of pectinolysis genes, which uses KDG as inducer. Thus, in Erwinia chrysanthemi, iron depletion or presence of KDG induces transcription of the genes involved in iron assimilation and pectinolysis. These important pathogenicity functions are coregulated by responding to common signals encountered in planta.