Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Summary

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Abstract

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The “last-line” antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

Introduction

The discovery of antibiotics is undoubtedly one of the most impactful health-related events of the 20^th century. Not only do antibiotics quickly resolve serious bacterial infections, they also play a pivotal role in modern medicine. Major surgeries, transplants and advances in neonatal medicine and chemotherapy leave patients susceptible to life threatening infections and these therapies would not be possible without antibiotics^1,2. However, rapid development and spread of antibiotic resistance among human pathogens has significantly decreased the efficacy of all clinically important classes of anti....

Protocol

1. Bacterial strain preparation

1. Streak the “donor” strain (E. coli MFD DAP^-/pJNW684, Table of Materials) for isolated colonies on Luria-Bertani agar supplemented with 600 μM diaminopimelic acid (DAP), 100 mg/L of ampicillin and 25 mg/L of kanamycin. Incubate overnight at 37 °C. Using a single isolated colony, inoculate 50 mL of Luria broth (LB) supplemented with 600 μM DAP, 100 mg/L of ampicillin and 25 mg/L of kanamycin in a 250 mL Erlenmeyer flask and label it as “donor”.
2. Streak the “recipient” strain (A. baumannii strain ATCC 17978, Table o....

Results

The outlined methods describe the generation of a high-density transposon library in A. baumannii strain ATCC 17978 through bacterial conjugation using E. coli MFD DAP^-, which replicates the plasmid pJNW684 (Figure 4B). The detailed protocol uses bi-parental bacterial conjugation for transfer of pJNW684 from the E. coli λpir+ donor strain to the A. baumannii recipient strain. This is an efficient and inexpensive method for generating de.......

Discussion

A. baumannii is an emerging threat to global public health due to the rapid acquisition of AMR against “last-line” therapeutics, such as colistin^{10,11,12,23,24,30,31}. In recent decades, Tn-seq has played a critical role in elucidating genotype-phenotype interactions acro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mM ddCTP, 2’,3’-Dideoxycytidine-5’-Triphosphate	Affymetrix	77112
100 mM dCTP 2’-Deoxycytidine-5’-Triphosphate	Invitrogen	10217-016
100bp DNA Ladder Molecular Weight Marker	Promega	PR-G2101
100mm x 15mm Petri Dishes	Corning	351029
150mm x 15mm Petri Dishes	Corning	351058
1X B&W	N/A	N/A	Dilute 2X B&W by half to get 1X B&W.
2,6-Diaminopimelic acid	Alfa Aesar	B2239103	used at 600 µM
2X B&W	N/A	N/A	Add 2 M NaCl, 10 mM Tris-HCl, 1 mM EDTA (pH 7.5) in water. Used with Streptavidin beads. Solutions keep at room temperature.
50mL Conical Sterile Polypropylene Centrifuge Tubes	Fisher Scientific	12-565-271
9.5 mM dCTP/0.5 mM ddCTP	N/A	N/A	9.5 ml 100 mM dCTP; 5 ml 10 mM ddCTP; 85.5 ml water. Store at -20°C.
AccuPrimeTM Pfx DNA Polymerase	Invitrogen	12344
Acinetobacter baumannii ATCC 17978	ATCC	N/A	AmpS, KanS
Ampicillin (100 mg/L)	Fisher Scientific	BP1760	used at 100 mg/L
AMPure XP PCR purification system	BECKMAN COULTER	A63881
BioAnalyzer	Agilent	G2939B
Bioanalyzer High Sensitivity DNA Analysis	Agilent	5067-4626
Deoxynucleotide Solution Mix (dNTP)	New England Biolabs (NEB)	N0447L
DynaMag-2 Magnetic rack	Invitrogen	12321D
E.coli MFD Dap-	N/A	N/A	DAP Auxotroph, requires 600 mM exogenously added DAP to grow. Contains RP4 machinery for plasmid transfer. Carrier for JNW68 (36).
Ethanol	Fisher Scientific	A4094
Externally Threaded Cryogenic Vials	Corning	09-761-71
Glass beads	Corning	72684
Glycerol	Fisher Scientific	G33
Inoculating loops	Fisher Scientific	22-363-602	Scraping tool
Kanamycin	Fisher Scientific	BP906	used at 25 mg/L
LB agar, Miller	Fisher Scientific	BP1425
LB broth, Miller	Fisher Scientific	BP1426
LoTE	N/A	N/A	Add 3 mM Tris-HCl, 0.2 mM EDTA (pH 7.5) in water. Used with Streptavidin beads. Solutions keep at room temperature.
Lysis buffer	N/A	N/A	9.34 mL TE buffer; 600 ml of 10% SDS; 60 ml of proteinase K (20 mg/mL)
Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.)	Fisher Scientific	BP1752I
Phosphate Buffered Saline, 10X Solution	Fisher Scientific	BP39920	Diluted to 1X
Qubit 4 Fluorometer	Thermo Fisher	Q33238
Qubit Assay Tubes	Thermo Fisher	Q32856
Qubit dsDNA HS Assay Kit	Thermo Fisher	Q32851
Sonicator with refridgerated waterbath	Qsonica Sonicators	Q2000FCE
Streptavidin Magnetic Beads	New England Biolabs (NEB)	S1420S
TE buffer	N/A	N/A	10 mM Tris-HCl (pH 8.0); 1 mM EDTA (pH 8.0)
Terminal Deoxynucleotidyl Transferase (rTdt)	Promega	PR-M1875