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Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution.

ABSTRACT: To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA. The activity of this clone was enhanced by 50 rounds of directed evolution by using DNA shuffling. Analysis of the evolved DNA fragments shows that two genetic regions have coevolved: One region, which has no obvious ORF, is essential for the activity, whereas the other, which appears to encode a protein, is not essential but enhances the activity of the former region. Analysis of the evolutionary intermediates shows that negative mutations are effectively removed while beneficial mutations accumulate and illustrates how a protein has evolved over the course of the evolution experiments. Although the mechanism of the activity remains unclear, the evolved DNA fragments also confer resistance to other drugs that inhibit bacterial cell-wall synthesis. The present system would serve as an experimental model to study evolutionary dynamics in the laboratory and provide the concept of screening natural libraries to obtain starting materials for directed evolution.

SUBMITTER: Yano T

PROVIDER: S-EPMC14682 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution.

Yano T T Kagamiyama H H

Proceedings of the National Academy of Sciences of the United States of America 20010123 3

To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA. The activity of this clone was enhanced by 50 rounds of directed evolution by using DNA shuffling. Analysis of the evolved DNA fragments shows that two genetic regions have coevolved: One region, which has no obvious ORF, is essential for the activity, whereas the other, which appears to encode a protein, is no ...[more]

PMID: 11158568

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Project description:Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 micro g/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 micro g/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 micro g/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 micro g/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 micro g/ml) lowered the ampicillin MIC to 3.67 micro g/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

Dataset Information

Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution.

Publications

Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution.

Similar Datasets

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