To explore whether transcription of cloned metagenomic DNA is interrupted by transcriptional terminators in environmental bacteria, we employed Substrate Induced Gene Expression (SIGEX) technology24. This method involves mixing cultures of bacterial strains that stably carry the same fosmid and then sorting for cells expressing GFP to identify functional metagenomic clones.

The bulk metagenomic library from a beach in Cadiz, Spain contaminated with crude oil was deposited on the SIGEX-compatible MPO555. The plasmid also contains an psal catabolic promoter that is commonly used to induce gene expression in cyanobacteria. A promoterless gfp gene that is downstream of the vector promoters and the metagenomic DNA cloning site was inserted in this plasmid, which allowed us to identify transcripts by detecting their GFP fluorescence and sorting based on this characteristic.

We constructed specialized E. coli strains MPO554 that produces the activator NahR and the antitermination protein N, and MPO555, similar to MPO554 but bearing a frameshift in gene N. The MPO555 strains were able to produce high levels of the T7 RNA polymerase in the presence of salicylate, a strong inhibitor of protein synthesis25. In the MPO555 strains, the lacUV5 promoter drives constitutive gene-1 transcription, and a nalidixic acid resistant derivative of the EPI300-T1 nitrate assimilator nasF promoter is under the control of the attenuator nasF to reduce expression vector transcription levels26.

In MPO555, psal-induced transcription of the gfp gene from pMPO579 and its derivative containing the thnL terminator pMPO580 was observed to be low. However, when MPO555 was induced with salicylate together with arabinose, the levels of gfp expression increased to a substantial extent. This suggests that the thnL terminator in pMPO580 does not completely terminate transcription from the psal promoter and indicates that production of NahR and N protein-dependent processive antitermination can restore transcription to a sufficient level for detection by GFP.

To test the ability of the MPO553 and MPO555 strains to transfer metagenomic clones by conjugation, we carried out triparental matings using EPI300-T1 carrying the bulk metagenomic library as donor, the rifampicin resistant (RifR) or nalidixic acid resistant (NalR) derivatives of this strain as recipient strains, and DH5a bearing pRK2013 as the helper strain. All of the recipient strains were able to transfer the metagenomic clones at frequencies greater than 6%, indicating that this specialized metagenomic pMPO579 vector is functional in different bacterial species. Despite the large amount of metagenomic DNA that each individual metagenomic clone is expected to contain, this high transfer rate indicates that the plasmid can efficiently mediate the transfer of metagenomic DNA between bacterial strains. This is in contrast to previous studies that have used broad host range vectors unable to support efficient mobilization of long stretches of environmental DNA27.