STUDY OF MN(II) OXIDATION MECHANISM AND ITS PHYSIOLOGICAL ROLE IN Pseudomonas resinovorans MOB-449

CIANCIO, L.; OTTADO, JORGELINA; GOTTIG, N.

Congreso; SAIB - SAMIGE Joint meeting 2021 on line; 2021

The environmental bacterium Pseudomonas resinovorans MOB-449 (MOB-449) was initially isolated by our group in order to analyze its use in Mn bioremediation processes. To this end, the MOB-449 strain was characterized according to its Mn(II) oxidation and biofilm formation capacities. These studies showed that MOB-449 is capable of oxidizing the metal only when it grows under static conditions and, unlike other Mn-oxidizing bacteria (MOB), it shows higher oxidation efficiency at 18°C than at 30°C (the optimal growth temperature). The focus of this work was to investigate the Mn(II) oxidation mechanism, especially at low temperatures. First of all, MOB-449 biofilm growth and development were analyzed in Lept medium, in the presence or absence of Mn(II). In both cases, a positive effect of the metal was detected at 18°C. This effect was accompanied by Manganese Oxide formation suggesting that the bacterium could obtain energy to grow through this process. Further, in vitro Mn(II) oxidase activity assays were performed with total protein extracts obtained at 18°C and different concentrations of uncoupling agent 2,4 Dinitrophenol (DNF). Results showed that the higher the concentration of DNF, the greater the Mn(II) oxidase activity, suggesting that Mn(II) oxidation could be a process that uses MOB-449 to obtain energy. In this context, and based on previous reports associating cytochromes with Mn(II) oxidation in other MOB, in silico searches of cytochrome terminal oxidase complexes present in MOB-449 sequenced genome were performed. MOB-449 genome denoted the presence of four cytochrome c terminal oxidases: the cbb3-1 oxidase (Cbb3-1), the cbb3-2 oxidase (Cbb3-2), the aa3 oxidase (Aa3), and the alternative-aa3 oxidase (Aa3). Specific oligonucleotides were designed to evaluate the expression levels of these four terminal oxidases by RT-qPCR. The results showed that genes analyzed that encode subunits of the four cytochrome c oxidases showed increased expression at 18 ºC in the presence of Mn(II). Subsequently, to found if specific cytochromes may oxidize Mn(II) delivering the electron to the cytochrome c terminal oxidase, the presence of cytochromes in total biofilm protein extracts of MOB-449 grown at 18 °C was determined. To this end, extracts obtained in the presence and absence of Mn(II) were separated by polyacrylamide gels electrophoresis and cytochromes were evidenced via their intrinsic peroxidase activity by using 3,3´,5,5´-Tetramethylbenzidine (TMBZ) and H2O2. In this way, a band of greater intensity was detected in the extracts with Mn(II) and the proteins present in this band will be identified by Mass Spectrometry. To conclude, the results obtained propose the Mn(II) oxidation as a form of chemolithotrophic metabolism of MOB-449 which could be vital for cellular functions at 18°C. However, future studies will be necessary to be able to elucidate with greater certainty the mechanism involved.