RESUMEN
YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M-1 s-1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.
Asunto(s)
Escherichia coli , Oxidorreductasas , Pseudomonas aeruginosa , Tiorredoxinas , Xylella , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/química , Filogenia , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/química , Xylella/enzimología , Xylella/genética , Xylella/metabolismoRESUMEN
Chalcogen bonds are the specific interactions involving group 16 elements as electrophilic sites. The role of chalcogen atoms as sticky sites in biomolecules is underappreciated, and the few available studies have mostly focused on S. Here, we carried out a statistical analysis over 3562 protein structures in the Protein Data Bank (PDB) containing 18â¯266 selenomethionines and found that Se···O chalcogen bonds are commonplace. These findings may help the future design of functional peptides and contribute to understanding the role of Se in nature.
Asunto(s)
Calcógenos/química , Fructoquinasas/química , Selenio/química , Aminoácidos/química , Cristalografía por Rayos X , Bases de Datos de Proteínas , Modelos Moleculares , Conformación Proteica , Selenometionina/química , Relación Estructura-Actividad , Xylella/enzimologíaRESUMEN
Xylella fastidiosa (Xf) is the xylem-dwelling bacterial agent associated with Pierce's disease (PD), which leads to significant declines in productivity in agriculturally important species like grapevine (Vitis vinifera). Xf spreads through the xylem network by digesting the pit membranes (PMs) between adjacent vessels, thereby potentially changing the hydraulic properties of the stem. However, the effects of Xf on water transport vary depending on the plant host and the infection stage, presenting diverse outcomes. Here, we investigated the effects of polygalacturonase, an enzyme known to be secreted by Xf when it produces biofilm on the PM surface, on stem hydraulic conductivity, and PM integrity. Experiments were performed on six grapevine genotypes with varying levels of PD resistance, with the expectation that PM resistance to degradation by polygalacturonase may play a role in PD resistance. Our objective was to study a single component of this pathosystem in isolation to better understand the mechanisms behind reported changes in hydraulics, thereby excluding the biological response of the plant to the presence of Xf in the vascular system. PM damage only occurred in stems perfused with polygalacturonase. Although the damaged PM area was small (2%-9% of the total pit aperture area), membrane digestion led to significant changes in the median air-seeding thresholds, and most importantly, shifted frequency distribution. Finally, enzyme perfusion also resulted in a universal reduction in stem hydraulic conductivity, suggesting the development of tyloses may not be the only contributing factor to reduced hydraulic conductivity in infected grapevine.
Asunto(s)
Proteínas de Insectos/metabolismo , Enfermedades de las Plantas , Poligalacturonasa/metabolismo , Vitis/fisiología , Xylella/fisiología , Xilema/fisiología , Resistencia a la Enfermedad , Membranas/fisiología , Tallos de la Planta/fisiología , Xylella/enzimologíaRESUMEN
The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals.
Asunto(s)
Cianuros/metabolismo , Glutarredoxinas/metabolismo , Estrés Oxidativo , Sulfurtransferasas/metabolismo , Xylella/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glutarredoxinas/genética , Modelos Moleculares , Filogenia , Conformación Proteica , Sulfurtransferasas/genética , Tiosulfato Azufretransferasa/metabolismoRESUMEN
Xylella fastidiosa is a gram-negative bacterium that causes Pierce's disease (PD) in grapevine. X. fastidiosa is xylem-limited and interfaces primarily with pit membranes (PMs) that separate xylem vessels from one another and from adjacent xylem parenchyma cells. PMs are composed of both pectic and cellulosic substrates, and dissolution of PMs is facilitated by X. fastidiosa cell wall-degrading enzymes. A polygalacturonase, which hydrolyzes the pectin component of PMs, is required for both movement and pathogenicity in grapevines. Here, we demonstrate that two X. fastidiosa ß-1,4-endoglucanases (EGases), EngXCA1 and EngXCA2, also play a role in how X. fastidiosa interfaces with grapevine PMs. The loss of EngXCA1 and EngXCA2 in tandem reduces both X. fastidiosa virulence and population size and slows the rate of PD symptom development and progression. Moreover, we demonstrate that single and double EGases mutants alter the rate of PD progression differently in two grapevine cultivars, Cabernet Sauvignon and Chardonnay, and that Chardonnay is significantly more susceptible to PD than Cabernet Sauvignon. Interestingly, we determined that there are quantitative differences in the amount of fucosylated xyloglucans that make up the surface of PMs in these cultivars. Fucosylated xyloglucans are targets of the X. fastidiosa EGases, and xyloglucan abundance could impact PM dissolution and affect PD symptom development. Taken together, these results indicate that X. fastidiosa EGases and the PM carbohydrate composition of different grape cultivars are important factors that influence PD symptom development and progression.
Asunto(s)
Celulasa , Vitis , Xylella , Celulasa/metabolismo , Enfermedades de las Plantas/microbiología , Especificidad de la Especie , Vitis/clasificación , Vitis/microbiología , Xylella/enzimologíaRESUMEN
Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.
Asunto(s)
Proteínas Bacterianas/química , Peróxido de Hidrógeno/química , Peroxidasas/química , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Xylella/enzimologíaRESUMEN
Isocitrate dehydrogenase (IDH) is a key enzyme at the critical junction between the tricarboxylic acid cycle and the glyoxylate cycle. Most bacteria have only one IDH, while a few contain two IDH isozymes. The coexistence of two different type IDHs in one organism was little known. Xylella fastidiosa is a nutritionally fastidious plant pathogen that contains two structurally different IDHs, an NAD+ -dependent homodimeric IDH (diXfIDH) and an NADP+ -dependent monomeric IDH (monoXfIDH). Kinetic characterization showed that diXfIDH displayed 206-fold preferences for NAD+ over NADP+ , while monoXfIDH showed 13,800-fold preferences for NADP+ over NAD+ . The putative coenzyme crucial amino acids (Asp-268, Ile-269, and Ala-275 in diXfIDH, and Lys-589, His-590, and Arg-601 in monoXfIDH) were studied by site-directed mutagenesis. The coenzyme specificities of the three diXfIDH mutants (D268K, D268K/I269Y, and D268K/I269Y/A275V) were switched successfully from NAD+ to NADP+ . Meanwhile, the mutant monoXfIDHs (H590L/R601L and K589T/H590L/R601L) greatly reduced the affinity for NADP+ , but failed to improve the ability to use NAD+ and had similar affinity to NADP+ and NAD+ . The biochemical properties of diXfIDH and monoXfIDH were investigated in detail. This study gives a further insight into the determinants of the coenzyme specificity in both monomeric and dimeric forms of IDHs.
Asunto(s)
Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/metabolismo , NADP/metabolismo , NAD/metabolismo , Xylella/enzimología , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Isocitrato Deshidrogenasa/genética , Cinética , Mutagénesis Sitio-Dirigida , Filogenia , Plantas/microbiología , Conformación Proteica , Multimerización de Proteína , Alineación de Secuencia , Especificidad por Sustrato , Temperatura , Xylella/química , Xylella/genética , Xylella/metabolismoRESUMEN
Xylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of its required insect vectors to ensure efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of the mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research, showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. Quantitative PCR assays allowed us to detect bacterial cells in the foregut of vectors after pathogen acquisition; populations of the wild-type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days, but not 3 days, post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but that cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to be paid to the substrates for this enzyme.
Asunto(s)
Quitina/metabolismo , Quitinasas/genética , Insectos/microbiología , Plantas/microbiología , Xylella/enzimología , Xylella/genética , Animales , Quitinasas/metabolismo , Insectos Vectores/microbiología , Enfermedades de las Plantas/microbiología , Xylella/patogenicidad , Xilema/microbiologíaRESUMEN
Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.
Asunto(s)
Carboxiliasas/química , Evolución Molecular , Lipopolisacáridos/química , Filogenia , Proteínas de Plantas/química , Xylella/genética , Secuencia de Aminoácidos , Secuencia de Bases , Carboxiliasas/genética , Carboxiliasas/metabolismo , Clonación Molecular , Coffea/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación del Sistema de Lectura , Expresión Génica , Hidrólisis , Lipopolisacáridos/biosíntesis , Monosacáridos/análisis , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Xylella/clasificación , Xylella/enzimología , Xylella/aislamiento & purificaciónRESUMEN
Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.
Asunto(s)
Proteínas Bacterianas/química , Calor , Glicoproteínas de Membrana/química , Ribonucleasas/química , Xylella/enzimología , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Glicoproteínas de Membrana/genética , Ribonucleasas/genética , Xylella/genética , Xylella/patogenicidadRESUMEN
In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/ß-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/ß -hydrolase fold.
Asunto(s)
Aldehído-Liasas/metabolismo , Esterasas/metabolismo , Xylella/enzimología , Aldehído-Liasas/química , Secuencia de Aminoácidos , Esterasas/química , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Xylella/químicaRESUMEN
Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4â Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31â Å. Two subunits form a molecular dimer in the asymmetric unit, which contains â¼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88â Å, ß=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains â¼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.
Asunto(s)
Proteínas Bacterianas/química , Glutamatos/química , Fosfotransferasas (aceptor de Grupo Carboxilo)/química , Xylella/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Glutamato Sintasa/química , Histidina , Enlace de Hidrógeno , Modelos Moleculares , Oligopéptidos , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Homología Estructural de ProteínaRESUMEN
Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Antibacterianos/farmacología , Comunicación Celular/fisiología , Fenotipo , Xylella/enzimología , Xylella/patogenicidad , Secuencia de Aminoácidos , Benzotiazoles , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Cartilla de ADN/genética , Diaminas , Resistencia a Medicamentos/genética , Escherichia coli , Eliminación de Gen , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Compuestos Orgánicos , Pseudomonas aeruginosa/enzimología , Quinolinas , Alineación de Secuencia , Tobramicina/farmacología , Vitis/microbiología , Xylella/efectos de los fármacos , Xylella/fisiologíaRESUMEN
The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity.
Asunto(s)
5'-Nucleotidasa/metabolismo , Xylella/enzimología , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/aislamiento & purificación , Biopelículas/crecimiento & desarrollo , Coenzimas/metabolismo , Perfilación de la Expresión Génica , Metales/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Xylella/fisiologíaRESUMEN
Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.
Asunto(s)
Proteínas Bacterianas/química , Proteasas de Cisteína/química , Modelos Moleculares , Xylella/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/agonistas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación Puntual , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Uridina Difosfato/química , Uridina Difosfato/metabolismoRESUMEN
Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.
Asunto(s)
Proteínas Bacterianas/química , Proteína Disulfuro Isomerasas/química , Multimerización de Proteína , Xylella/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Cobre/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Enfermedades de las Plantas/microbiología , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Estructura Cuaternaria de Proteína , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Difracción de Rayos X , Xylella/genética , Xylella/fisiologíaRESUMEN
Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK toxin-antitoxin (TA) system. PemK toxin inhibits bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK by direct binding. PemK and PemI were overexpressed in Escherichia coli and activities of each were assessed. Purified PemK toxin specifically degraded single-stranded RNA but not double-stranded RNA, double-stranded DNA, or single-stranded DNA. Addition of PemI antitoxin inhibited nuclease activity of PemK toxin. Purified complexes of PemI bound to PemK exhibited minimal nuclease activity; removal of PemI antitoxin from the complex restored nuclease activity of PemK toxin. Sequencing of 5' rapid amplification of cDNA ends products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU and UACG. Nine single amino-acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, ribonuclease activity, and PemI binding. Three PemK point-substitution mutants (R3A, G16E, and D79V) that lacked nuclease activity did not inhibit growth. All nine PemK mutants retained the ability to bind PemI. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the R100 pemI/pemK TA system of E. coli.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endorribonucleasas/metabolismo , ARN Bacteriano/metabolismo , Xylella/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Endorribonucleasas/genética , Endorribonucleasas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mutación , Plásmidos , Mapeo de Interacción de Proteínas , ARN Bacteriano/genética , Proteínas Recombinantes de Fusión , Especificidad por Sustrato , Xylella/genéticaRESUMEN
Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized.
Asunto(s)
Proteínas Bacterianas/química , Manosiltransferasas/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Xylella/enzimología , Proteínas Bacterianas/genética , Enzimas Inmovilizadas/química , Manosiltransferasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Xylella/genéticaRESUMEN
The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Peroxidasas/metabolismo , Proteínas Represoras/metabolismo , Xylella/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Lipoilación , Modelos Biológicos , Oxidación-Reducción , Unión Proteica , Especificidad por SustratoRESUMEN
The transformation efficiency of Xylella fastidiosa can be increased by interfering with restriction by the strain-specific type II system encoded by the PD1607 and PD1608 genes. Here, we report results for two strategies: in vitro methylation using M.SssI and isolation of DNA from an Escherichia coli strain expressing the methylase PD1607.