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ACS Infect Dis. 2018 Jun 8;4(6):1007-1018. doi: 10.1021/acsinfecdis.8b00017. Epub 2018 Mar 23.

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

Author information

1
Department of Microbiology and Immunobiology , Harvard Medical School , 4 Blackfan Circle , Boston , Massachusetts 02115 , United States.
2
Department of Chemistry and Chemical Biology , Harvard University , 12 Oxford Street , Cambridge , Massachusetts 02138 , United States.

Abstract

Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

KEYWORDS:

bacterial genetics; chemical genetics; homologous recombination; negative selection; pathway inhibitors

PMID:
29534563
PMCID:
PMC5993608
[Available on 2019-06-08]
DOI:
10.1021/acsinfecdis.8b00017

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