We have constructed a genetically compatible system that allows the transfer of metagenomic DNA fragments from rifampicin resistant (RifR) and nalidixic acid resistant (NalR) derivatives of E. coli to non-adapted host strains with very high efficiency. This system takes advantage of the specialized transcription systems of these host strains and their innate resistance to b-lactam antibiotics. This strategy was used to select for fosmids containing a gene encoding the components of an efflux pump that resist carbenicillin (Cb). Among 100 CbR transconjugants isolated from EPI300-T1 NalR derivatives carrying the bulk metagenomic library, we identified 6 distinct fosmids that conferred resistance to Cb. All of these fosmids had Orfs coding for the three components of an efflux pump that are induced by nalidixic acid.

The plasmid pCC1FOS-CeuI contains the lacUV5 promoter regulatory sequence, the attenuator nasF and gene-1 of the T7 phage coding for the T7 RNA polymerase. To allow the T7 RNA polymerase to be expressed from this vector in a non-hosting strain, we constructed the MPO555 strain with the nahGHILNJK operon promoter, the NahR activator and the lambda phage N antitermination protein inserted into its trg locus (Fig. 1; for MPO553 construction details see supplementary Methods). This insertion also results in expression of the thnL terminator in the host strain.

Using this system, we cloned the metagenomic DNA from an environmental sample collected from the shore of Punta San Garcia in Cadiz contaminated by crude oil from a tanker spill. The metagenomic DNA was transcribed by the T7 RNA polymerase from a vector containing the T7 gene 10 promoter, and its synthesis was terminated at the thnL terminator by the induction of nahR by salicylate in the host strain. Expression of the gfp gene was then monitored by fluorescent microscopy, revealing that transcription from the psal promoter is activated by NahR in a non-hosting strain and that synthesis of the N protein is able to impose processive anti-termination on nahR-induced transcripts.

We then tested the ability of this system to transfer metagenomic clones from the donor host to non-hosting strains by conjugation. To this end, triparental matings were performed with the rifampicin resistant and nalidixic acid resistant MPO553 and MPO555 carrying the bulk metagenomic library as donors, each of the above specialized host strains bearing pCC1FOS-CeuI and the metagenomic clones, and DH5a containing pRK2013 as the helper strain. We found that pMPO579 was transferred to all recipient strains with a very high conjugation frequency of over 10%, similar to that obtained with the mobilizable plasmid pBBR1MCS-3.