MPO555

 

The ability to efficiently clone metagenomic DNA and to identify active clones is a critical step towards the effective utilization of the extraordinary biodiversity of uncultivated bacteria. However, a significant limitation of current functional metagenomic screening systems is the inability to detect complex regulatory systems by identifying their transcriptional activators or anti-termination proteins. The MPO555 system overcomes this limitation by integrating a promoterless GFP gene downstream of vector and metagenomic DNA cloning sites. This allows for exploitation of Substrate Induced Gene Expression (SIGEX) technology24 to identify regulatory systems by detection of GFP fluorescence, and by subsequent phage transcriptional anti-termination protein N-dependent processing of the cloned metagenomic DNA.

To develop the MPO555 system, a DNA fragment containing the lacUV5 promoter, the attenuator nasF and the thnL terminator of the lambda phage gene N was inserted into the E. coli EPI300-T1 strain at its trg locus, to yield pMPO571. This was then combined with the metagenomic DNA cloning site and the T7 gene 10 promoter to yield pMPO579. An additional modification of pMPO571 to yield pMPO580 involved cloning the thnL terminator upstream of a strong ribosome binding site from the T7 gene 10. The resulting system has the potential to identify regulatory systems by detection of T7 RNA polymerase-dependent gfp gene transcription in vivo.

Induction of a large number of transconjugants resistant to 100 mg L-1 carbenicillin (CbR) by the MPO555 system was demonstrated. The specialized MPO555 strains MPO554 and MPO555, which contain the metagenomic DNA cloning fragment and the activator NahR in their trg loci respectively, showed a 6-fold increase in CbR resistant transconjugants compared to the conventional EPI300-T1 NalR strain.

Detailed analysis of MPO555 strains showed that the psal promoter in pMPO580 is able to activate transcription from metagenomic DNA and that the thnL terminator can be evaded by NahR-dependent processive anti-termination mediated by a truncated lambda phage N gene. However, induction of gfp expression from psal in the presence of arabinose and salicylate was very low. Increasing the plasmid copy number resulted in a modest increase in gfp expression, but high levels of gfp expression were obtained when arabinose and salicylate were added together. This demonstrated that the thnL termination signal is not essential for activation of gfp transcription in this system and that production of NahR and N can significantly improve the performance of the MPO555 system.