Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation.

A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422 750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.

An expanded CRISPR-Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates.

Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.