Resumo

The main reservoir of antibiotic resistance genes (ARG) is the soil. The environmental diversity of ARG needs closer attention – as so, metagenomic libraries constructed with soil samples has been screened for the identification of ARG. It has been proposed that ARG, as well as antibiotics, play distinct roles in nature when compared to the clinical context. Hence, the identification of new resistance genes from soil and other samples could reveal both the real functions of these genes in the environment as well as potential elements with biotechnological applications. Small and large-insert Cerrado soil metagenomic libraries were screened with 9 β-lactamic antibiotics. In total, 62 resistant clones were isolated to 5 of the 9 antibiotics, in the 2 libraries. Two clones were analyzed by ORFinder and Blastp and 2 ORF were identified: AMX3(3) and CRB2(1). CRB2(1) was also analyzed with PSIPRED3.0 for secondary structure prediction. Blastp runs revealed 2 genes related to the metabolism of aromatic compounds – both are dioxygenases and were named AMX3(3) and CRB2(1), similar to intradiol ring-cleavage dioxygenases and gentisate dioxygenases, respectively. However, both hits have low scores and similarity indexes. This result strongly suggests the identification of a new dioxygenase family. As gentisate dioxygenases carry a characteristic secondary structure (cupin module), a prediction and comparison of CRB2(1) and a characterized gentisate dioxygenase structure was performed. Both have similar structures composed by a bicupin module. The screening of a soil metagenomic library suggests that the theory that the primary roles of ARG in nature are diverse from the clinical context is plausible. The analysis of 2 ORFs identified in two resistant metagenomic clones showed similarity to dioxygenases. In terms of resistance, this could mean that either the bacteria use the antibiotic as a carbon source or the structure of the antibiotics are similar to the enzymatic substrate, showing a functional shift and a resistant phenotype. This is observation is crucial, as these genes could become potential ARG in the clinical environment. These are versatile enzymes, capable of degrading different aromatic by-products and are of high biotechnological value in bioremediation.