IDENTIFICATION OF GENES INVOLVED IN MN(II) OXIDATION PROCESS IN Pseudomonas resinovorans STRAIN MOB-513

PARRA, L; PIAZZA, AINELÉN; OTTADO, JORGELINA; GOTTIG, N.

Congreso; SAMIGE 2022; 2022

The presence of soluble manganese Mn(II) affects the quality of groundwater, a source of drinking water for many populations, and is an important environmental concern. Biological sand filter technology, based on bacterial oxidation of metals to form insoluble oxides that can be filtered out of the water, is widely used for groundwater potabilization. Bioaugmentation of biological sand filters with Mn(II)-oxidizing bacteria (MOB) is used to increase Mn removal efficiencies from groundwater. The environmental isolate Pseudomonas resinovorans strain MOB-513 improves Mn groundwater removal. Interestingly, previous studies showed that this bacterium can oxidize Mn(II) only in the biofilm lifestyle and that c-di-GMP, a second messenger crucially involved in Pseudomonas biofilm formation, increases biofilm-formation and Mn(II)-oxidizing capabilities in MOB-513. In addition, it was determined that this novel role for c-di-GMP in the up-regulation of Mn(II) oxidation is performed through induction of the expression of Manganese-Oxidizing Peroxidase (MOP) enzymes. In order to identify genes involved in MOP expression and additional regulatory factors of Mn(II) oxidation, in this work, a transposon (Tn) mutagenesis was performed in MOB-513. The pALMAR-3 plasmid harboring the Tn5 was introduced into MOB-513 by electroporation and the transformants were selected in Lept-Mn medium, especially used for Mn(II) oxidation, supplemented with tetracicline 12,5 μg/ml. In this medium the clones that oxidize Mn(II) can be visualized by the appearance of a brown color due to Mn oxides. A total of 30.000 transformants were obtained and 428 were white indicating the lack of Mn(II) oxidation in these clones, on the other hand 284 clones showed a higher capacity of Mn(II) oxidation than MOB-513 wild type. For these mutant clones two sets of PCR primers were used to amplify the DNA flanking the transposon insertion performing colony PCR assays. In PCR round 1, an arbitrary primer (ARB-1B), and TnExt ALMAR3-PCR (which is complementary to the transposon sequence) were used and for the PCR round 2 (that enhance the amount of the specific product), the arbitrary primer ARB-1 and TnInt ALMAR3-seq were used. The PCR products were purified and sequenced using the Tnlnt primer in LMBA-FCByF-UNR. This strategy allows the identification of several genes that encode transcription factors, two component system response regulators and proteins involved in biofilm formation. Future studies with these mutants may allow to determine the specific roles of the mutated genes in Mn(II) oxidation and also if they have a link with c-di-GMP regulatory network.