Meta-omic evaluation of bacterial microbial community structure and activity for the environmental assessment of soils: overcoming protein extraction pitfalls.

Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris-HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC-MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism.