Aconitase B Is Required for Optimal Growth of Xanthomonas campestris pv. vesicatoria in Pepper Plants

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcUAAAH) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the ΔhrcU mutant with HrcUAAAH produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the ΔhrcU mutant complemented with HrcUAAAH, suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the ΔhrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta

Nuclear Magnetic Resonance metabolomics reveals an excretory metabolic signature of renal cell carcinoma

RCC usually develops and progresses asymptomatically and, when detected, it is frequently at advanced stages and metastatic, entailing a dismal prognosis. Therefore, there is an obvious demand for new strategies enabling an earlier diagnosis. The importance of metabolic rearrangements for carcinogenesis unlocked a new approach for cancer research, catalyzing the increased use of metabolomics. The present study aimed the NMR metabolic profiling of RCC in urine samples from a cohort of RCC patients (n = 42) and controls (n = 49). The methodology entailed variable selection of the spectra in tandem with multivariate analysis and validation procedures. The retrieval of a disease signature was preceded by a systematic evaluation of the impacts of subject age, gender, BMI, and smoking habits. The impact of confounders on the urine metabolomics profile of this population is residual compared to that of RCC. A 32-metabolite/resonance signature descriptive of RCC was unveiled, successfully distinguishing RCC patients from controls in principal component analysis. This work demonstrates the value of a systematic metabolomics workflow for the identification of robust urinary metabolic biomarkers of RCC. Future studies should entail the validation of the 32-metabolite/resonance signature found for RCC in independent cohorts, as well as biological validation of the putative hypotheses advanced