Functional screening of soil metagenomic libraries

Most of the genomes of environmental microorganisms are inaccessible because they cannot be cultured in the lab by standard techniques. However, we can access the genomes of these unculturable organisms by extracting DNA directly from environmental samples and creating metagenomic DNA libraries. Using this approach, DNA from thousands of environmental microbes can be functionally screened for a variety of abilities. We are pursuing 2 different strategies to create and functionally screen environmental DNA libraries.

Using standard methods to functionally screen DNA libraries in an E. coli host, we are investigating the antibiotic resistance mechanisms coded in uncultured soil microorganisms. In particular, we have identified sequences from an environmental DNA library that allow growth of an E. coli host in the presence of 6 different antibiotics that function by targeting varied cellular pathways. We have found new sequences coding for many families of antibiotic resistance proteins including the antibiotic modifying enzymes rifampin ADP-ribosylases and aminoglycoside acetyltransferases, transporter proteins that are able to pump antibiotics out of the cell, as well as proteins like dihydrofolate reductases that are able to evade antibiotics when exogenously expressed in E. coli. We hope to use these new sequences to learn more about the evolution and functions of these protein families.

We are additionally interested in developing new ways to screen environmental DNA libraries in order to overcome the limitations associated with functional screening in a laboratory host. Standard screening requires that a heterologously expressed protein is functional in the host bacteria, and also requires the availability of an assay to test the function of interest on a large scale. Cloning an environmental DNA library into a phage backbone and screening the phage library via affinity selection would allow more permissive and efficient screening since protein domains that bind to a substrate of interest could be recovered without relying on the function of the encoded protein in a foreign host. This screening strategy will be widely applicable to a variety of binding and catalytic functions. We hope to use affinity selection of a metagenomic phage display library to search for antibiotic resistance proteins and inhibitors of these resistance proteins.

Figure 2. Antibiotic resistance profiling of a soil metagenomic library. A. Number of resistant clones recovered against each antibiotic. A library of 1.4e06 clones with an average insert size of 1.5 kb was screened against 6 antibiotics. A total of 41 resistant clones have been identified. B. Distribution of amino acid identities for 41 resistance genes recovered from soil samples compared to the most similar gene from any organism in GenBank.