RESUMEN
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice. As a part of its virulence repertoire, Xoo secretes a cell wall degrading enzyme Cellobiosidase (CbsA), which is a critical virulence factor and also a determinant of tissue specificity. CbsA protein is made up of an N-terminal catalytic domain and a C-terminal fibronectin type III domain. According to the CAZy classification, the catalytic domain of CbsA protein belongs to the glycosyl hydrolase-6 (GH6) family that performs acid-base catalysis. However, the identity of the catalytic acid and the catalytic base of CbsA is not known. Based on the available structural and biochemical data, we identified putative catalytic residues and probed them by site-directed mutagenesis. Intriguingly, the biochemical analysis showed that none of the mutations abolishes the catalytic activity of CbsA, an observation that is contrary to other GH6 family members. All the mutants exhibited altered enzymatic activity and caused significant virulence deficiency in Xoo emphasising the requirement of specific exoglucanase activity of wild-type CbsA for virulence on rice. Our study highlights the need for further studies and the detailed characterisation of bacterial exoglucanases.
Asunto(s)
Oryza , Xanthomonas , Virulencia/genética , Oryza/metabolismo , Dominio Catalítico , Xanthomonas/genética , Xanthomonas/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
As the most abundant biopolymer on earth, cellulose undergoes degradation by a diverse set of enzymes with varying specificities that act in synergism. An assay protocol was developed to detect and quantify activity of cellulose 1,4-ß-cellobiosidase (EC 3.2.1.91) in soil. The optimum pH and temperature for ß-cellobiosidase activity were approximately pH 5.5 and 60 °C, respectively. In the tested six soils, the Michaelis constants (Km) ranged from 0.08 to 0.51 mM, and maximum velocity (Vmax) ranged from 71.5 to 318.1 µmol kg soil-1 h-1. The temperature coefficient (Q10) ranged from 1.72 to 1.99 at non-denaturing temperatures from 10 to 50 °C, and the activation energy (Ea) ranged from 42.5 to 53.7 kJ mol-1. The assay procedure provided reproducible results with a coefficient of variance ≤4.7% and demonstrated a limit of quantification (LOQ) of 50.9 µmol p-nitrophenol release kg-1 soil h-1 for ß-cellobiosidase activity in soil. Notably, the developed assay protocol offers reproducibility and precision comparable to bench-scale assays while reducing costs associated with reagents, supplies, and labor.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa , Celulosa , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Reproducibilidad de los Resultados , Temperatura , Suelo , CinéticaRESUMEN
Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Asunto(s)
Xanthomonas campestris , Xanthomonas , Genómica , Especificidad de Órganos , Señales de Clasificación de Proteína , Xanthomonas/genética , Xanthomonas campestris/genéticaRESUMEN
BACKGROUND Because of the insensitivity of renal cell carcinoma (RCC) to both chemotherapy and radiotherapy, surgery remains the primary approach for anticancer treatment. However, patients who do not receive timely diagnoses may not be suitable for surgery, especially in the late phase of tumor development. Thus, the discovery of novel effective treatment is of great importance. Allyl isothiocyanate (AITC) can inhibit the proliferation and induce apoptosis in many cancer cells. In this paper, we report on an in vitro study to determine the effect of AITC on proliferation and apoptosis of RCC line GRC-1. MATERIAL AND METHODS CCK8 assay was used to detect cell proliferation under gradient concentrations of AITC. Flow cytometry was employed to evaluate cell apoptosis. Real-time fluorescent polymerase chain reaction quantified mRNA levels of Bax and Bcl-2 genes. Western blotting was further employed for protein expression assay. RESULTS AITC inhibited GRC-1 cell proliferation and induced cell apoptosis in a dose-dependent manner; it also elevated Bax while suppressing Bcl-2 gene expression at both mRNA and protein levels. In general, increasing concentration of AITC decreased Bcl-2/Bax ratio. CONCLUSIONS The inhibitory effect of AITC on GRC-1 cells is exerted via cell apoptosis, in which the imbalance of Bcl-2/Bax plays a significant role.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Isotiocianatos/farmacología , Neoplasias Renales/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteína X Asociada a bcl-2/biosíntesis , Adulto , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Genes bcl-2/efectos de los fármacos , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 µM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co(2+), Cu(2+), Ni(2+) and Mn(2+) (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-ß-cellobiosidase that hydrolyzes cellulose into cellobiose.