Approaches for diversity analysis of cultivable and non-cultivable bacteriain real soil

Until recently, investigators had no idea how accurately cultivated microorganisms represented the overall microbial diversity. The cultivation-dependent approach is limited by the fact that the overwhelming majority of microorganisms present in soil cannot be cultivated under laboratory conditions. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. There is a vast amount of information held within the genomes of cultivable and non-cultivable microorganisms, and new methods based on analysis of DNA allow to investigate this potential. In this work we show some aspects, advantages and disadvantages of classical and new approaches in taxonomical and functional description of bacteria present in natural microbial assemblages on the example of cultivable bacteria isolated from rhizosphere of plants, tobacco and black nightshade, planted in PCB contaminated soil. Biochemical analysis of isolates showed 8 different bacterial species. This identification was compared by discrimination using MALDI-TOF mass spectrometry and identity evaluation after sequencing of 16S rDNA. Six strains from original number of 8 were positively identified after 16S rDNA sequencing and their phylogenetic relations were compared. These analyses confirmed closed relations of all species (two of isolates exhibited the same characteristics and were discriminated as the same species Pseudomonas stutzeri) and also of Burkholderia xenovorans LB 400, a well-known PCB degrader. Nevertheless, only two isolates gave a positive reaction after amplification of the biphenyl dioxygenase gene and exhibited potential to degrade PCB. These results indicate that only a subset of the recovered molecular information, derived from active population based on molecular and functional analysis is relevant to microbial ecology.