Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins

Abstract

To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibiotic-resistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a beta-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional beta-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a beta-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

FIG. 1.

Detection of strA and strB streptomycin phosphotransferases in the metagenomic library. Each of five subpools of the metagenomic library, as well as a positive and a negative control, was used as a template to amplify the strA and strB genes. A total of 5 μl of each PCR was separated on a 1% agarose gel in 1× Tris-borate-EDTA (TBE). Lane M contains the 1-kb DNA ladder (Promega, Madison, WI). Primers specific to strA were used in lanes labeled “strA” and should result in a band at 753 bp. Primers specific to strB were used in lanes labeled “strB” and should result in a band at 540 bp. Lanes labeled “+” are positive-control PCRs using the strA and strB genes as templates. Lanes labeled “−” contain products of PCR with no DNA template as a negative control. The sizes of the molecular weight markers are indicated on the left.

FIG. 2.

The domains of Cft2. (A) The cft2 ORF is predicted to encode a bifunctional protein. The N-terminal domain, amino acids 1 to 211, encodes a predicted ECF-type sigma factor. The C-terminal domain, amino acids 343 to 607, encodes a class A β-lactamase. (B) The β-lactamase domain of cft2 was cloned into pET28b and expressed in E. coli. The numbers above each ORF reflect amino acid numbering. MIC values are for the full-length and truncated cft2 constructs.

FIG. 3.

Clones conferring kanamycin resistance. (A) The kan4 ORF is predicted to encode a bifunctional protein. The N-terminal domain is predicted to be an aminoglycoside acetyltransferase. The C-terminal domain encodes a predicted GCN5-related acetyltransferase. Proteins encoded by AOKan6 and AOKan8 were aligned to Kan4. The percentages represent the pairwise amino acid identities from each alignment. (B) The parts of kan4 encoding the two domains were cloned separately into pET44a and expressed in E. coli. The numbers above each ORF reflect amino acid numbering. The MICs represent the levels of resistance to kanamycin conferred by each of the clones.