Characterization of the Genetic Potential, Catabolic Structure and Degradative Activities against BTEX in Microbial Communities from Aquifers under Adaptation to Organic Contaminants

Abstract

Developments in molecular techniques have led to rapid and reliable tools to monitor microbial community structures and dynamics under in situ conditions. However, even though various functional genes are localized on mobile genetic elements such that metabolic potential/activity is not necessarily reflected by the community structure, there has been a lack of emphasis on monitoring functional diversity. A more detailed picture of the catabolic gene structure and sequence diversity in environmental samples will significantly increase our knowledge of the functional potential of microbial communities. We adapted suitable techniques to follow functional gene diversity and applied those to target catechol 2,3-dioxygenases as key genes in aromatic hydrocarbon degradation. Catabolic gene diversity in differentially BTEX contaminated environments was assessed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs. The PCR-SSCP technique could be shown to be a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the biodegradation functioning of microbial communities. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish pre-selection or enrichment bias and to assess the function of predominant gene polymorphs. To rapidly determine phylogenetic diversity of functional genes from strain collections or environmental DNA amplifications, a restriction enzyme, theoretically producing characteristic profiles, the similarities of which reassembled the main divergent branches of C23O gene phylogeny, was used to perform an amplified functional DNA restriction analysis (AFDRA) on C23O genes of reference strains and isolates. Sequences of PCR fragments from isolates were in close agreement with the phylogenetic correlations predicted with the AFDRA approach. AFDRA thus provided a quick assessment of C23O diversity in a strain collection and insights of its gene phylogeny affiliation. AFDRA was also used to determine the predominant polymorphism of the C23O gene present in environmental DNA extracts and in combination with a most-probable-number-PCR approach, its abundance. This approach may be useful to differentiate functional genes also for many other gene families. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralising phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralise benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. Complete C23O encoding open reading frames were cloned, sequenced and overexpressed by using conserved regions in operon neighbouring genes. Such strategy also allows the direct access to complete genes from environmental DNA. A single amino acid substitution at position 218 had severe influence on enzyme kinetics, and the Tyr218 variant differed from the His218 variant by lower turnover number but higher affinity. The information in this work underlines the importance to analyze catabolic gene diversity at different scales, from global views of diversity and fitness of genes in the ecosystem, to detailed understanding of sequence variations effects on catalytic activities.