Development and application of in situ PCR techniques to investigate microbial community functional and phylogenetic diversity at the individual cell level

Abstract

We developed a number of in situ PCR techniques using model gene systems in laboratory-grown strains of bacteria, and then modified these techniques for use with natural bacterial communities. Depending on the particular technique used, we could target DNA, mRNA, or rRNA to examine either functional genes or gene activity or as a means to examine phylogenetic relationships amongst bacterial communities. We also applied these newly developed technologies to a pilot study in which we examined the response of both low-impact and high-impact microcosm communities to toluene. We found no influence of toluene on changes in bacterial cell number, nor on the numbers of BTEX-degraders as measured by in situ PCR. However, in high-impact communities, we observed a marked increase in total bacterial cell number as well as the number of BTEX degraders within the communities.