23 - Biodegradación, Biorremediación y Biodeterioro

ANALYSIS OF THE MOLECULAR MECHANISMS OF Mn(II) OXIDATION IN Pseudomonas resinovorans TO ENHANCE METAL BIOREMEDIATION

Gaffuri, Marilina¹ - Parra, Lucía¹ - Ottado, Jorgelina² - Gottig, Natalia¹

1) Instituto de Procesos Biotecnológicos y Químicos de Rosario (IPROBYQ-CONICET-UNR) Rosario-Santa Fe-Argentina
2) Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET-UNR) Rosario-Santa Fe-Argentina
Contacto: gaffurimarilina@gmail.com

Groundwater is a crucial drinking water source, but in many areas of Argentina, it contains unacceptable levels of manganese (Mn) in the Mn(II) state. Biological sand filtration is an efficient and eco-friendly method to purify this water and Manganese-Oxidizing Bacteria (MOB) accelerates Mn(II) removal. Previously, we isolated and characterized various MOB, selecting those with high adherence efficiency, biofilm formation, and Mn(II) oxidation capabilities. In one of the isolates, Pseudomonas resinovorans MOB-513, the intracellular levels of cyclic dimeric GMP (c-di-GMP) were enhanced by the overexpression of a diguanylate cyclase (DGC), which synthesize this messenger. Interestingly, we found that c-di-GMP enhance both biofilm formation and Mn(II) oxidation capacity. To investigate the role of c-di-GMP in Mn(II), MOB-513, transposon mutants that lost their ability to oxidize Mn(II) were selected. We found mutants in genes of Type IV Pili (Δpil) involved in bacterial twitching motility, and mutants in the hk-2948 (Δhk) gene which encodes a sensor histidine kinase with unknown function. In the upstream region, a gene that encodes a response regulator with a DGC domain, rr-2947 (rr), is present. To characterize these mutants twitching mobility, biofilm formation and macrocolony Mn(II) oxidation assays were performed. As expected, Δpil mutants did not display twitching motility, but only ΔpilC and ΔpilH lost their ability to form biofilm compared to WT strain. Neither ΔpilC+DGC nor ΔpilH+DGC recovered the ability to form biofilm. Mn(II) oxidation assays on macrocolonies showed that only ΔpilH+DGC recovered the ability to oxidize Mn(II). This suggests that both PilC and elevated levels of c-di-GMP are crucial for Mn(II) oxidation. Δhk strain exhibited similar twitching motility than WT strain but showed a significant reduction in biofilm formation. We could not complement the Δhk mutant or overexpress HK-2948 in the WT strain, may be as a consequence of the large size of the protein, or difficulties in its insertion into the membrane. Nevertheless, a Δhk+DGC strain was constructed and it regained the ability to form biofilms and to oxidize Mn(II) comparted to Δhk-pEmpty. These results, suggest a role of HK-2948 in the increase of c-di-GMP necessary for biofilm and Mn(II) oxidation process. RR-2947 protein levels were increased in WT and Δhk strains by the overexpression of its gene using the pBBR1 vector (WT+rr and Δhk+rr strains). In WT+rr, an increase in biofilm formation and earlier Mn(II) oxidation were observed compared to WT+pEmpty, and similar to WT+DGC, indicating the involvement of this RR in these process. However, Δhk+rr did not shown enhanced biofilm formation and Mn(II) oxidation. These results suggest that HK-2948 is required to activate RR-2947, leading to increased c-di-GMP levels and, consequently, biofilm formation and Mn(II) oxidation. In conclusion, this study suggests that Type IV Pili, HK-2948 and RR-2947 are essential for Mn(II) oxidation.

Palabras clave: Pseudomonas resinovorans – Manganese – Biofilm - c-di-GMP – Oxidizing Bacteria