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Department of Plant Pathology and Microbiology
The Robert H. Smith Faculty of Agriculture, Food & Environment
The Hebrew University of Jerusalem

P.O. Box 12 
Rehovot 76100 
ISRAEL

Tel: 08-9489219
Fax: 08-9466794
rakefetk@savion.huji.ac.il

Publications

2018
Kviatkovski, I. ; Yarnitzky, T. ; Shushan, S. ; Schwartz-Harari, O. ; Nir-Paz, R. ; Helman, Y. A bacterial biosensor encoding a genetically modified LuxR receptor exhibits improved detection of Pseudomonas aeruginosa's biomarker molecule 2-aminoacetophenone. Chemical Communications 2018, 54, 9218 - 9221. Publisher's VersionAbstract
2-Aminoacetophneone (2-AA) is a volatile molecule produced in high amounts by the opportunistic pathogen Pseudomonas aeruginosa. We have previously shown that 2-AA activates the quorum sensing (QS) LuxR receptor of Aliivibrio fischeri. In the present study we were able to improve LuxR's affinity and detection limit for 2-AA by genetic modification of three amino acids within the binding pocket of the receptor. Expression of the modified LuxR receptor in a luminescent bacterial biosensor provided an efficient detection assay of 2-AA in clinical P. aeruginosa strains isolated from blood and lung infections, as well as in phlegm samples obtained from subjects suffering from lung infections.
Kviatkovski, I. ; Shushan, S. ; Oron, Y. ; Frumin, I. ; Amir, D. ; Secundo, L. ; Livne, E. ; Weissbrod, A. ; Sobel, N. ; Helman, Y. Smelling Pseudomonas aeruginosa infections using a whole-cell biosensor - An alternative for the gold-standard culturing assay. J Biotechnol 2018, 267, 45-49.Abstract
Improved easy-to-use diagnostic tools for infections are in strong demand worldwide. Yet, despite dramatic advances in diagnostic technologies, the gold-standard remains culturing. Here we offer an alternative tool demonstrating that a bacterial biosensor can efficiently detect Pseudomonas aeruginosa infections in patients suffering from otitis externa. Detection was based on specific binding between the biosensor and 2-aminoacetophenone (2-AA), a volatile produced by P. aeruginosa in high amounts. We collected pus samples from ears of 26 subjects exhibiting symptoms of otitis externa. Detection of P. aeruginosa using the biosensor was compared to detection using gold-standard culturing assay and to gas-chromatograph-mass-spectrometry (GC-MS) analyses of 2-AA. The biosensor strain test matched the culture assay in 24 samples (92%) and the GC-MS analyses in 25 samples (96%). With this result in hand, we designed a device containing a whole-cell luminescent biosensor combined with a photo-multiplier tube. This device allowed detection of 2-AA at levels as low as 2 nmol, on par with detection level of GC-MS. The results of the described study demonstrate that the volatile 2-AA serves as an effective biomarker for P. aeruginosa in ear infections, and that activation of the biosensor strain by 2-AA provides a unique opportunity to design an easy-to-use device that can specifically detect P. aeruginosa infections.
2016
Shavit, R. ; Lebendiker, M. ; Pasternak, Z. ; Burdman, S. ; Helman, Y. The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module. Frontiers in Microbiology 2016, 6 1499. Publisher's VersionAbstract
Toxin–antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin’s activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin–antitoxin encoding system in the genome of the plant pathogen Acidovorax citrulli. The system is homologous to vapB–vapC systems from other bacterial species. PCR and phylogenetic analyses suggested that this locus is unique to group II strains of A. citrulli. Using biochemical and molecular analyses we show that A. citrulli VapBC module is a bona-fide toxin–antitoxin module in which VapC is a toxin with ribonuclease activity that can be counteracted by its cognate VapB antitoxin. We further show that transcription of the A. citrulli vapBC locus is induced by amino acid starvation, chloramphenicol and during plant infection. Due to the possible role of TA systems in both virulence and dormancy of human pathogenic bacteria, studies of these systems are gaining a lot of attention. Conversely, studies characterizing toxin–antitoxin systems in plant pathogenic bacteria are lacking. The study presented here validates the activity of VapB and VapC proteins in A. citrulli and suggests their involvement in stress response and host–pathogen interactions.