BACTERIAL TRANSCRIPTIONAL FACTOR-BASED BIOSENSOR FOR DETECTING PHOSPHONATES

MASOTTI, F.; KRINK, N.; LENCINA, N; GOTTIG, N.; NIKEL, P.; OTTADO, JORGELINA

Congreso; XVIII Congreso Samige; 2023

More than 50,000 ton of phosphonates (Phn) are used annually, representing a 3% increase over the last decade. Their highly stable chemical structure and global demand make Phns an abundant pollutant with political, health and environmental implications. Glyphosate (GP) herbicide is part of these compound groups and the major worldwide agrochemical used for weed control. Recently, in our laboratory we identified a bacterial strain, Agrobacterium tumefaciens CHLDO (hereafter CHLDO) that was able to degrade GP as phosphorous source, and genome sequencing indicated that GP degradation could be performed by means of a C-P lyase pathway codified in a phn cluster. This cluster consists of 15 transcriptional units (phnFGHIJKLO-duf1045-phnMN cluster) that encompass a transcriptional regulator (PhnF), the C-P lyase complex (PhnGHIJKL), an aminoalkylphosphonate N-acetyl-transferase (PhnO), a transport system (PhnDCE1E2), and CP lyase accessory proteins (duf1045-PhnMN). In other bacteria, the phn cluster is controlled by a dual regulatory mechanism: an upregulation under conditions of phosphate starvation by the PhoR/PhoB two component signalling system and PhnF-mediated repression.The CHLDO-phn cluster contains six intergenic regions that could be considered as putative promoters: PphnG, PphnJ, PphnC, PphnD, PphnE2 and Pduf1045. In-silico analysis showed that PphnG, PphnC, and Pduf1045 are endowed with P-responsive PhoB boxes as have been described as promoters in other Agrobacterium-related bacteria. Also, PphnG contains a PhnF binding box. By studying the native promoter architecture, six fluorescence-dependent plasmids were constructed using Synthetic Biology tools and introduced in Agrobacterium spp. When these strains were incubated in a minimal medium supplemented with Phns as phosphorous source, PphnG derivative biosensor was found to be induced with a modest sensibility. Further, this biosensor was re-engineered and an extra-copy of phnF coding for the regulator has been introduced. The resulting biosensor was able to sense GP in a detection range of 85 – 4,200 ppb, with higher sensibility and dynamic range than the precursor. However, this biosensor is still not specific for GP due to the fact that could sense other Phn such as aminomethyl phosphonic acid (AMPA). Finally, this biosensor could be applied to detect toxic Phns such as GP and AMPA from complex samples such as herbicide-contaminated soil and compared to results obtained by other detection methods, such as HPLC. In summary, the toolkits and methodologies developed during this work opens up avenues for both fundamental and applied research.