RESUMEN
Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.
Asunto(s)
Proteínas Bacterianas/química , Modelos Moleculares , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Benzoatos/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ácido Sórbico/análogos & derivados , Ácido Sórbico/química , Ácido Sórbico/metabolismo , Ácido Sórbico/farmacología , Xylella/genética , Xylella/metabolismo , Xylella/fisiologíaRESUMEN
The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Proteínas Represoras , Xylella/genética , Secuencia de Bases , Dicroismo Circular , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Plantas/metabolismo , Plantas/microbiología , Regiones Promotoras Genéticas/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia , Transcripción Genética/fisiología , Xylella/metabolismo , Xylella/patogenicidadRESUMEN
The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.