RESUMEN
Bacteria, fungi, and mammals contain lactonases that can degrade the Gram-negative bacterial quorum sensing (QS) molecules N-acyl homoserine lactones (AHLs). AHLs are critical for bacteria to coordinate gene expression and pathogenicity with population density. However, AHL-degrading lactonases present variable substrate ranges, including degradation of the Pencillium expansum lactone mycotoxin patulin. We selected Erwinia spp. as our model bacteria to further investigate this interaction. We find both native apple microbiome Erwinia spp. and the fruit tree pathogen Erwinia amylovora to be inhibited by patulin. At patulin concentrations that inhibited E. amylovora growth, expression of E. amylovora lactonase encoded by EaaiiA was increased. EaAiiA demonstrated the ability to degrade patulin in vitro, as well, as in vivo where it reduced apple disease and patulin production by P. expansum. Fungal-bacterial co-cultures revealed that the E. amylovora Δeaaiia strain failed to protect apples from P. expansum infections, which contained significant amounts of patulin. Our results suggest that bacterial lactonase production can modulate the pathogenicity of P. expansum in response to the secretion of toxic patulin. IMPORTANCE: Chemical signaling in the microbial world facilitates the regulation of gene expression as a function of cell population density. This is especially true for the Gram-negative bacterial signal N-acyl homoserine lactone (AHL). Lactonases that deactivate AHLs have attracted a lot of attention because of their antibacterial potential. However, the involvement of these enzymes in inhibiting fungal pathogens and the potential role of these enzymes in bacterial-fungal interactions are unknown. Here, we find that a bacterial enzyme involved in the degradation of AHLs is also induced by and degrades the fungal lactone mycotoxin, patulin. This work supports the potential use of bacterial enzymes and/or the producing bacteria in controlling the post-harvest fruit disease caused by the patulin-producing fungus Penicillium expansum.
Asunto(s)
Hidrolasas de Éster Carboxílico , Erwinia amylovora , Malus , Patulina , Patulina/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/genética , Malus/microbiología , Erwinia amylovora/genética , Erwinia amylovora/efectos de los fármacos , Erwinia amylovora/enzimología , Erwinia amylovora/metabolismo , Enfermedades de las Plantas/microbiología , Penicillium/genética , Penicillium/enzimología , Penicillium/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Interacciones Microbianas , Percepción de Quorum , Lactonas/metabolismo , Lactonas/farmacologíaRESUMEN
AIM: Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells. METHODS AND RESULTS: Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a Ki of 3·7 µmol l-1 . An E. amylovora â¿ghxP::Camr strain shows resistance to growth on 8AG and 6TG. Although EaGhxP is expressed during active disease propagation, it is not necessary for virulence as determined on immature apple and pear assays. CONCLUSIONS: EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.
Asunto(s)
Erwinia amylovora/metabolismo , Guanina/metabolismo , Hipoxantina/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Tioguanina/metabolismo , Azaguanina/metabolismo , Erwinia amylovora/enzimología , Erwinia amylovora/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Malus/microbiología , Proteínas de Transporte de Nucleobases/genética , Enfermedades de las Plantas/microbiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Here, two kinds of polysaccharide-based biocomposites were investigated. The enzymatically synthesized levan from Erwinia amylovora was applied as the matrix, while montmorillonite clay and bovine serum albumin (BSA) were used as additive in the biocomposite. To examine the properties of levan/MMT biocomposite, we choose different ratios between levan and MMT to implement the surface morphology observation, thermal property analysis, and rheological behavior determination. As a result, the levan/MMT biocomposite in a 2:1 blending ratio showed a significant improvement both in the thermal and rheological properties. Meanwhile, the 0.1 % levan/BSA nanoparticle showed the highest encapsulation capacity and surface charge as 53.13⯱â¯2.64 % and +3.92⯱â¯0.43â¯mV. Last but not least, the levan/BSA nanoparticle exhibited a slower and controlled release of the BSA from the system. All of these results indicated a potential application of levan-based biocomposite and nanoparticle.
Asunto(s)
Bentonita/química , Materiales Biocompatibles/química , Fructanos/química , Nanopartículas/química , Albúmina Sérica Bovina/química , Animales , Materiales Biocompatibles/síntesis química , Bovinos , Erwinia amylovora/enzimología , Fructanos/biosíntesis , Hexosiltransferasas/metabolismo , Tamaño de la Partícula , Proteínas Recombinantes/metabolismo , Propiedades de SuperficieRESUMEN
The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2EA) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malusâ¯×â¯robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2EA, a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85â¯Å. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Erwinia amylovora/metabolismo , Malus/microbiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Cristalografía por Rayos X , Erwinia amylovora/enzimología , Interacciones Huésped-Patógeno , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the ß-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or ß-lysylated EF-P.IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.
Asunto(s)
Proteínas Bacterianas/genética , Erwinia amylovora/genética , Regulación Bacteriana de la Expresión Génica , Hidroliasas/genética , Factores de Elongación de Péptidos/genética , ARN Helicasas/genética , Proteínas Bacterianas/metabolismo , Erwinia amylovora/enzimología , Erwinia amylovora/patogenicidad , Eliminación de Gen , Prueba de Complementación Genética , Hidroliasas/metabolismo , Lisina/metabolismo , Malus/microbiología , Mutación , Factores de Elongación de Péptidos/deficiencia , Fenotipo , Enfermedades de las Plantas/microbiología , Plásmidos/química , Plásmidos/metabolismo , ARN Helicasas/deficiencia , VirulenciaRESUMEN
Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics.IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.
Asunto(s)
Sistemas de Transporte de Aminoácidos/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Erwinia amylovora/efectos de los fármacos , Erwinia amylovora/enzimología , Glicina/análogos & derivados , Análisis Mutacional de ADN , Farmacorresistencia Bacteriana , Glicina/farmacología , MutaciónRESUMEN
Erwinia tasmaniensis is an epiphytic bacterium related to the plant pathogen Erwinia amylovora, the etiological agent of fire blight. In this study the levansucrase from E. tasmaniensis (EtLsc) has been compared with the homologous enzyme from E. amylovora (EaLsc). We characterized the enzymatic activity and compared the products profile of both enzymes by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detector (HPAEC-PAD). Moreover we determined the crystal structure of EtLsc to understand the structural peculiarity causing the different product profiles of the two homologues. EtLsc exhibits increased efficiency in the production of FOS, resulting in a better catalyst for biotechnological synthesis than EaLsc. Based on our results, we propose that the role of this enzyme in the life cycle of the two bacteria is most likely related to survival, rather than linked to pathogenicity in E. amylovora.
Asunto(s)
Proteínas Bacterianas , Erwinia amylovora , Hexosiltransferasas , Análisis de Secuencia de ADN , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Erwinia amylovora/enzimología , Erwinia amylovora/genética , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Enfermedades de las Plantas/microbiologíaRESUMEN
Less attention has been focused on the industrial applications of levan-type fructan than that of inulin. Levan-type fructan is a unique homopolysaccharide consisting of fructose residues with a ß-(2, 6) linkage that possesses unique physiochemical properties such as low intrinsic viscosity. In this study, the recombinant levansucrase from Erwinia amylovora was used to efficiently produce levan from sucrose, and under optimised conditions, 195â¯g/L levan was produced from 500â¯g/L sucrose, with the highest conversion rate of 59%. The physicochemical properties of E. amylovora levan, such as surface morphology, thermal behaviour, rheology behaviour and texture analysis, were evaluated and compared with those of commercial gels, including xanthan, guar, carrageenan and Arabic gums. The produced E. amylovora levan showed a series of acceptable physicochemical properties, indicating a potential application for levan as a novel water-soluble micro gel. The conclusions of this study support the exploration of the use of more hydrogels in the food, medicinal and cosmetic industries.
Asunto(s)
Erwinia amylovora/química , Fructanos/química , Agua/química , Erwinia amylovora/enzimología , Erwinia amylovora/metabolismo , Industria de Alimentos , Fructanos/biosíntesis , Geles , Hexosiltransferasas/metabolismo , Peso Molecular , Solubilidad , TemperaturaRESUMEN
6-Thioguanine (6TG) is a DNA-targeting therapeutic used in the treatment of various cancers. While 6TG was rationally designed as a proof of concept for antimetabolite therapy, it is also a rare thioamide-bearing bacterial natural product and critical virulence factor of Erwinia amylovorans, plant pathogens that cause fire blight. Through gene expression, biochemical assays, and mutational analyses, we identified a specialized bipartite enzyme system, consisting of an ATP-dependent sulfur transferase (YcfA) and a sulfur-mobilizing enzyme (YcfC), that is responsible for the peculiar oxygen-by-sulfur substitution found in the biosynthesis of 6TG. Mechanistic and phylogenetic studies revealed that YcfA-mediated 6TG biosynthesis evolved from ancient tRNA modifications that support translational fidelity. The successful inâ vitro reconstitution of 6TG thioamidation showed that YcfA employs a specialized sulfur shuttle that markedly differs from universal RNA-related systems. This study sheds light on underexplored enzymatic C-S bond formation in natural product biosynthesis.
Asunto(s)
Antimetabolitos/metabolismo , Proteínas Bacterianas/metabolismo , Erwinia amylovora/enzimología , Tioamidas/metabolismo , Tioguanina/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas , Erwinia amylovora/genética , Erwinia amylovora/metabolismo , Oxígeno/metabolismo , Filogenia , Enfermedades de las Plantas/microbiología , Transducción de Señal , Azufre/metabolismoRESUMEN
Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84â¯Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Erwinia amylovora/enzimología , Erwinia amylovora/metabolismo , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Proteínas Bacterianas/genética , Erwinia amylovora/genética , Hexosafosfatos/metabolismo , Cinética , Rosaceae/microbiología , Deshidrogenasas del Alcohol de Azúcar/genética , Tomografía Computarizada por Rayos XRESUMEN
Extensive use of the antibiotic streptomycin to control fire blight disease of apples and pears, caused by the enterobacterial plant pathogen Erwinia amylovora, leads to the development of streptomycin-resistant strains in the United States and elsewhere. Kasugamycin (Ksg) has been permitted to be used as an alternative or replacement to control this serious bacterial disease. In this study, we investigated the role of two major peptide ATP-binding cassette transporter systems in E. amylovora, the dipeptide permease (Dpp) and oligopeptide permease (Opp), in conferring sensitivity to Ksg and blasticidin S (BcS). Minimum inhibitory concentration and spot dilution assays showed that the dpp deletion mutants exhibited slightly enhanced resistance to Ksg in rich medium, whereas the opp mutant exhibited slightly enhanced resistance to Ksg in minimal medium and BcS in rich medium. Deletion of both dpp and opp conferred a higher level of resistance to Ksg in both rich and minimal media, whereas deletion of opp alone was sufficient to confer high level of resistance to BcS in minimal medium. In addition, bioinformatic analysis combined with reverse transcription-quantitative polymerase chain reaction showed that the Rcs phosphorelay system negatively regulates opp expression and the rcsB mutant was more sensitive to both Ksg and BcS in minimal medium as compared with the wild type. An electrophoresis motility shift assay further confirmed the direct binding of the RcsA/RcsB proteins to the promoter region of the opp operon. However, neither the Dpp nor the Opp permeases contributed to disease progress on immature pears, hypersensitive response on tobacco leaves, or exopolysaccharide amylovoran production. These results suggested that Ksg and BcS employ the Dpp and Opp permeases to enter E. amylovora cells and the Dpp and Opp permeases act synergistically for illicit transport of antibiotics.
Asunto(s)
Aminoglicósidos/farmacología , Erwinia amylovora/efectos de los fármacos , Erwinia amylovora/genética , Proteínas de Transporte de Membrana/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Erwinia amylovora/enzimología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genoma Bacteriano , Mutación , Nucleósidos/farmacologíaRESUMEN
The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H2 O2 ) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H2 O2 -neutralizing enzymes, such as catalases, during plant-pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non-host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H2 O2 and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant-pathogen interactions; (iii) the induction of EPSs by H2 O2 despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation-induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non-culturable (VBNC) response.
Asunto(s)
Catalasa/metabolismo , Erwinia amylovora/enzimología , Erwinia amylovora/patogenicidad , Factores de Virulencia/metabolismo , Catalasa/genética , Erwinia amylovora/efectos de los fármacos , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/farmacología , Espectrometría de Masas , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Enfermedades de las Plantas/microbiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c-di-GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c-di-GMP-dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Celulosa/biosíntesis , GMP Cíclico/análogos & derivados , Erwinia amylovora/patogenicidad , Factores de Virulencia/metabolismo , Biopelículas/efectos de los fármacos , Celulosa/metabolismo , GMP Cíclico/farmacología , Erwinia amylovora/enzimología , Erwinia amylovora/genética , Erwinia amylovora/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Operón/genética , VirulenciaRESUMEN
Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.
Asunto(s)
Proteínas Bacterianas/química , Erwinia amylovora/enzimología , Glucofosfatos/química , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Uridina Difosfato Glucosa/química , Uridina Trifosfato/química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Erwinia amylovora/química , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosamina/análogos & derivados , Galactosamina/química , Galactosamina/metabolismo , Galactosafosfatos/química , Galactosafosfatos/metabolismo , Expresión Génica , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , Glucofosfatos/metabolismo , Cinética , Manosafosfatos/química , Manosafosfatos/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pentosafosfatos/química , Pentosafosfatos/metabolismo , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/química , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
AmsI is a low-molecular-weight protein tyrosine phosphatase that regulates the production of amylovoran in the Gram-negative bacterium Erwinia amylovora, a specific pathogen of rosaceous plants such as apple, pear and quince. Amylovoran is an exopolysaccharide that is necessary for successful infection. In order to shed light on AmsI, its structure was solved at 1.57â Å resolution at the same pH as its highest measured activity (pH 5.5). In the active site, a water molecule, bridging between the catalytic Arg15 and the reaction-product analogue sulfate, might be representative of the water molecule attacking the phospho-cysteine intermediate in the second step of the reaction mechanism.
Asunto(s)
Arginina/química , Proteínas Bacterianas/química , Cisteína/química , Erwinia amylovora/química , Polisacáridos Bacterianos/química , Proteínas Tirosina Fosfatasas/química , Secuencia de Aminoácidos , Arginina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Cisteína/metabolismo , Erwinia amylovora/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Plásmidos/química , Plásmidos/metabolismo , Polisacáridos Bacterianos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Agua/química , Agua/metabolismoRESUMEN
Many bacteria produce isonitrile-containing natural products that are derived from aromatic amino acids. The synthetic clusters that control biosynthesis most commonly encode two enzymes, designated PvcA and PvcB, as well as additional enzymes that direct synthesis of the natural product. The PvcA enzyme installs the isonitrile moiety at the amino group of either tyrosine or tryptophan, as dictated by the particular pathway. The common pathway intermediate produced by PvcA is directed toward different ultimate products by PvcB, a member of the family of Fe(2+), α-ketoglutarate-dependent oxygenases. To continue our investigation of the structural and functional properties of the isonitrile biosynthetic pathways, we present here a study of the PvcB homologues from three organisms. Two pathways, derived from Pseudomonas aeruginosa and Xenorhabdus nematophila, produce known products. A third PvcB homologue from Erwinia amylovora is part of an uncharacterized pathway. Our results demonstrate the diversity of reactions catalyzed. Although all PvcB enzymes catalyze the hydroxylation of the tyrosine isonitrile substrate, the elimination of the hydroxyl in Pseudomonas and Erwinia is driven by deprotonation at Cα, resulting in the initial production of an unsaturated tyrosine isonitrile product that then cyclizes to a coumarin derivative. PvcB from Xenorhabdus, in contrast, catalyzes the same oxygenation, but loss of the hydroxyl group is accompanied by decarboxylation of the intermediate. Steady-state kinetic analysis of the three reactions and a docking model for the binding of the tyrosine isonitrile substrate in the PvcB active site highlight subtle differences between the PvcB homologues.
Asunto(s)
Proteínas Bacterianas/química , Erwinia amylovora/enzimología , Oxigenasas/química , Pseudomonas aeruginosa/enzimología , Xenorhabdus/enzimología , Proteínas Bacterianas/metabolismo , Oxigenasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4"-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4"-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.
Asunto(s)
Erwinia amylovora/metabolismo , Azúcares de Uridina Difosfato/química , Azúcares de Uridina Difosfato/metabolismo , Conformación de Carbohidratos , Difosfatos/química , Erwinia amylovora/enzimología , Fosfotransferasas/metabolismo , UDPglucosa 4-Epimerasa/metabolismoRESUMEN
Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.
Asunto(s)
Proteínas Bacterianas/fisiología , Erwinia amylovora/patogenicidad , Enfermedades de las Plantas/microbiología , Factor sigma/fisiología , Proteínas Bacterianas/genética , Eriobotrya/microbiología , Erwinia amylovora/enzimología , Erwinia amylovora/genética , Genes Bacterianos , Respuesta al Choque Térmico/genética , Hexosiltransferasas/metabolismo , Mutación , Presión Osmótica , Estrés Oxidativo/genética , Polisacáridos Bacterianos/metabolismo , Pyrus/microbiología , Rosaceae/microbiología , Factor sigma/genética , Virulencia/genéticaRESUMEN
Glucose-1-phosphate uridylyltransferase from Erwinia amylovora CFPB1430 was expressed as a His-tag fusion protein in Escherichia coli. After tag removal, the purified protein was crystallized from 100â mM Tris pH 8.5, 2â M ammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46â Å using synchrotron radiation. The crystals belonged to the hexagonal space group P62, with unit-cell parameters a = 80.67, b = 80.67, c = 169.18. The structure was solved by molecular replacement using the structure of the E. coli enzyme as a search model.
Asunto(s)
Cristalografía por Rayos X/métodos , Erwinia amylovora/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Reacción en Cadena de la Polimerasa , Conformación Proteica , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/aislamiento & purificaciónRESUMEN
Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.