Diversity of the gene of benzoate dioxygenase in bacterial associations isolated from long term organochlorine-contaminated soils

Abstract

Background. Communities of bacteria with specific enzymes are formed in the soil with long-term organochlorine contamination. The aim of this study was to analyze the diversity of the benA gene encoding the -subunit of the benzoate 1,2-dioxygenase in aerobic bacterial associations isolated from the soils of the Chapayevsk-city (Samara region, Russia). Materials and methods. The soil samples were taken on the territory, contaminated with organochlorine compounds for a long time. As a selection factor in the enrichment cultures were used 4-chlorobenzoic acid and chlorobenzene, in the pure cultures benzoic acid. The isolation of total DNA from bacterial associations was performed using a commercial FastDNA Spin Kit for Soil kit (USA). Amplification was performed on a MyCycler instrument (USA). Determination of the nucleotide sequence was performed on an automatic sequencer Genetic Analyzer 3500XL (USA). The search and analysis for benA gene homologs was carried out using international GenBank databases and BLAST system (http://www.ncbi.nlm.nih.gov). Results. As a result of selection, 12 associations of aerobic bacteria were obtained. Fragments of the benA gene (-subunit of benzoate dioxygenase) were obtained with the total DNA of six bacterial associations selected on chlorobenzene and with the total DNA of three bacterial associations selected on 4-chlorobenzoate. Pure cultures of aerobic bacterial strains using benzoic acid as a carbon source were isolated from benA-positive associations. It was established that the amplified fragments with the DNA of the A1, A4, A5, B1, B2, B3, B4 and B6 association strains form a single phylogenetic cluster with the -subunit gene of the benzoate dioxygenase of the Pseudomonas putida strain KT2440 (level of similarity is 9698%). The amplified fragment with the DNA of strain B5-170 (association B5) forms a cluster with the gene of the -subunit of the benzoate dioxygenase of the strain Pseudomonas sp. VLB120 (93% similarity).