Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Summary

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Abstract

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This "evolutionary rescue" approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Introduction

Antibiotic resistance in bacteria is a major health problem¹. At a fundamental level, the spread of antibiotic resistance in pathogenic bacteria is a simple example of evolution by natural selection^2,3. Put simply, the use of antibiotics generates selection for resistant strains. A key problem in evolutionary biology, therefore, is to understand the factors that influence the ability of bacterial populations to evolve resistance to antibiotics. Selection experiments have emerged as a very powerful tool to investigate the evolutionary biology of bacteria, and this field has produced incr....

Protocol

1. Construction of the Experimental System Encoding Antibiotic Resistance Gene

Note: Here E. coli MG1655 was used as the recipient strain of the plasmid- or chromosome-encoded antibiotic resistance gene. The antibiotic resistance gene is encoded in the chromosome or a multicopy plasmid in an otherwise isogenic strain (Figure 1).

1. Insertion of the antibiotic resistance gene in the λ phage integration site (attB)²⁰ of the chromosome of MG1655.
   1. Amplify 500 bp-long regions at both sides of the chromosomal attB site by PC....

Results

In our previous work, the evolution the β-lactamase gene bla_TEM-1 towards conferring resistance to the third generation cephalosporin ceftazidime¹² was investigated. This gene was selected because, although TEM-1 does not confer resistance to ceftazidime, mutations in bla_TEM-1 can expand the range of activity of TEM-1 to hydrolyze cephalosporins such as ceftazidime²⁹. Mutations in antibiotic resis.......

Discussion

We present a new protocol combining molecular biology, experimental evolution and deep DNA sequencing designed to investigate the role of multicopy plasmids in the evolution of antibiotic resistance in bacteria. Although this protocol combines techniques from different fields, all the methods required to develop it are simple, and can be performed in a regular microbiology laboratory. The most critical steps in the protocol probably are the construction of the model system strains and the reconstruction of the mutations .......

Materials

Name	Company	Catalog Number	Comments
Thermocycler	BioRad	C1000
Electroporator	BiorRad	1652660
Electroporation cuvettes	Sigma-Aldrich	Z706078
NanoDrop 2000/2000c	Thermo Fisher Scientific	ND-2000	Determine DNA quality measuring the ratios of absorbance 260nm/280nm and 260nm/230nm
Incubator	Memmert	UF1060
Incubator (shaker)	Cole-Parmer Ltd	SI500
Electrophoresis power supply	BioRad	1645070	Agarose gel electrophoresis
Electrophoresis chamber	BioRad	1704405	Agarose gel electrophoresis
Pippettes	Biohit	725020, 725050, 725060, 725070
Multi-channel pippetes	Biohit	728220, 728230, 728240
Plate reader Synergy HTX	BioTek	BTS1LF
Inoculating loops	Sigma-Aldrich	I8388
96-well plates	Falcon	351172
LB	BD Difco	DF0446-17-3
LB agar	Fisher scientific	BP1425-500
Phusion Polymerase	Thermo Fisher Scientific	F533S
Gibson Assembly	New England Biolabs	E2611S
Resctriction enzymes	Fermentas FastDigest
Antibiotics	Sigma-Aldrich
QIAprep Spin Miniprep Kit	Qiagen	27104	Plasmid extraction kit
Wizard Genomic DNA Purification Kit	Promega	A1120	gDNA extraction kit
DNeasy Blood & Tissue Kits	Qiagen	69506	gDNA extraction kit
Electroporation cuvettes	Sigma-Aldrich	Z706078
Petri dishes	Sigma-Aldrich	D9054
Cryotubes	ClearLine	390701
96-well plates (-80ºC storage)	Thermo Fisher Scientific	249945
QuantiFluor dsDNA System	Promega	E2670	Quantification of DNA concentartion
Agarose	BioRad	1613100	Agarose gel electrophoresis
50x TAE buffer	BioRad	1610743	Agarose gel electrophoresis
T4 Polynucleotide Kinase	Thermo Fisher Scientific	EK0031
T4 DNA Ligase	Thermo Fisher Scientific	EL0014