Development of a novel ribosomal RNA based method for fingerprinting bacterial consortia

Abstract

The last two decades have seen a revolution in the development of molecular based methods to investigate microbial diversity and function, such as stable isotope probing (SIP) methods and fluorescence in situ hybridisation-based technology. Unprecedented advancements in our knowledge have been achieved largely thanks to the advent of the polymerase chain reaction (PCR), which is the basis for many current microbial fingerprinting techniques and clone libraries. A major drawback of PCR is that accurate information of species abundance cannot be gained, because of procedural biases which substantially modify abundance information. Additionally, in SIP studies the isotopic signal is erased after DNA amplification. Consequently, the option of quantifying isotopic enrichment directly from specific taxa is lost.

This work describes the development of a novel method that eliminates the need of PCR in microbial community fingerprinting. It is based on the separation of small rRNA fragments by polyacrylamide gel electrophoresis on the basis of sequence-dependant conformational differences. The rRNA fragments are created by a targeted cleavage of the ribosomal small subunit using Ribonuclease H. The development and optimisation of the protocol to achieve this is presented here. The method was tested with several environmental samples and showed that it had greatest utility when applied to samples harbouring low microbial diversity. This was confirmed through profiling of several artificial communities. Taxonomic identification of bacterial species represented by bands in profiles was determined, as well as their relative abundance. The method was applied to follow changes in community diversity and abundance in three different types of enrichment cultures. Experiments were also carried out to determine the isotopic content of [13]C-labelled rRNA retrieved from acrylamide gels and the detection of [14]C-labelled RNA from profile bands. It was shown that removal of contaminating [12]C from [13]C-RNA was essential for detection of isotopic signatures and that radioactive rRNA bands can be detected by phosphor imaging technology. Overall, the method termed here ‘Direct rRNA Fingerprinting’ is a PCR-free alternative that offers detection of microbial abundance and activity using radioactive or stable isotope labelling. Further methodological developments that address its limitations will enhance its sensitivity and utility in diversity and functional studies.