Getting closer to tehe understanding of the copper-resistance mechanisms in Apiotrichum loubieri M12

Abstract

The extreme conditions present in acid mine drainage (AMD)-affected environments promote the adaptation of the native microbiota to that adverse conditions. For these reason, these environments constitute interest niches for the isolation of heavy metal-resistant microorganisms. In our previous works, Apiotrichum loubieri M12 was isolated from sediments of the AMD-affected environment located in La Carolina (San Luis, Argentina). The microorganism was able to tolerate and remove Cu(II) from liquid culture media, reaching a 30-35% removal capacity when it was exposed to 40 µg mL-1 Cu(II) after 48 h of incubation. Likewise, in the presence of copper, the intracellular protein expression was clearly affected. Taking into account these preliminary results, the present work proposes both the extracellular proteomic analysis and the study of the microelemental composition and surface mapping of the microbial biomass, in the presence and absence of Cu(II), to in-depth into the understanding of the removal and tolerance mechanisms of A. loubieri M12 when exposed to copper. To achieve this purpose, Scanning Electron Microscopy coupled to X-Ray Dispersive Energy Spectrometry (SEM-EDS) was applied to analyze the microelemental composition and the surface mapping of the microbial biomass, in the presence and absence of 40 µg mL-1 Cu(II) after 48 h of incubation at 200 rpm and 30 °C. Additionally, a shotgun proteomic analysis was carried out on the 20X-concentrated cell-free supernatants to analyze the differential protein expression at the extracellular level in the presence of the metal. SEM-EDS results indicated a uniform bioadsorption of the metal on the cell surface, found at 4.09% w/w, and variations in the proportion of other biomass´ constituent elements. A four-fold decrease in K intensity and the peaks corresponding to N and Mg were detected only in the biomass exposed to the metal. When analyzing the extracellular proteomic results, we detected proteins capable to sequester bivalent ions. Interestingly, a specific response to the copper presence was detected in the cell-free supernatants of A. loubieri M12, where proteins involved in the transport of copper ions into and/or out of the cell were identified. The results obtained in this study guide us to conclude that the electronegative elements of the cell wall play an important role in the uptake of the copper ions. The exposure to the metal may also cause ion exchange mechanisms on the cell surface. Likewise, the differential expression of proteins in the extracellular space is crucial for the sequestration and transport of the metal, fundamental to reduce the toxic effects that Cu(II) could exert on the cell.