Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

Abstract

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Figure 1. Schematic diagram of the fosmids derived from pCC1FOS-CeuI.

Modifications resulting from this work, including oriT, psal, the nut_L site and the promoterless gfp shown in the amplified region.

Figure 2. Schematic diagram of the specialized strains derived from E. coli EPI300™-T1, showing the DNA fragment that has been integrated into their trg locus.

Figure 3. Expression of the gfp gene from pMPO579, or its derivative pMPO580 bearing the thnL terminator, in the strains MPO554 that produces the activator NahR and the antitermination protein N, and MPO555 that is similar to MPO554 but bears a frameshift in the gene N.

Figure 4. Expression of the gfp gene from pMPO579 or its derivative bearing the thnL terminator pMPO580, in the strain MPO553 that constitutively produces low levels of T7 RNA-polymerase.

Figure 5. Schematic diagram showing the clones that confer carbenicillin resistance.

All putative orfs and their orientations are shown although only those putatively involved in carbenicillin resistance are highlighted. Genes are not drawn to scale.