Diversity of ring-hydroxylating dioxygenases in pristine and oil contaminated microbial mats at genomic and transcriptomic levels

The aim of this work was to characterize bacterial ring-hydroxylating dioxygenase (RHD) diversity in a pristine microbial mat and follow their diversity changes in response to heavy fuel oil contamination. In order to describe the RHDs diversity, new degenerate primers were designed and a nested-PCR approach was developed to gain sensitivity and wider diversity. RHD diversity in artificially contaminated mats maintained in microcosms and in chronically contaminated mats was analysed by clone libraries and terminal restriction fragment length polymorphism (T-RFLP) at genomic and transcriptomic levels. The RHD diversity in the pristine microbial mat was represented by Pseudomonas putida nahAc-like genes and no increase of diversity was detected after 1 year of oil contamination. The diversity observed in a 30 year chronically polluted microbial mat was represented by four main RHD clusters and two new genes revealing higher polyaromatic hydrocarbon (PAH) degradation capacity. This study illustrates that a single petroleum contamination (such as oil spill) is not enough to involve a detectable modification of RHD diversity. The new degenerate primers described here allowed RHD gene amplification from pristine and contaminated samples thereby showing their diversity. The proposed approach solves one of the main problems of functional gene analysis providing effective amplification of the environmental diversity of the targeted genes.