Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Summary

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using φBB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Abstract

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage φBB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage φBB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Introduction

The development of tools for the genetic manipulation of the spirochetal bacterium Borrelia burgdorferi has added immeasurable value to the understanding of the nature of Lyme disease^1,2,3,4. B. burgdorferi has an unusually complex genome comprised of a small linear chromosome and both linear and circular plasmids^5,6. Spontaneous plasmid loss, intragenic rearrangement (movement of genes from one plasmid to another within the same organism), and horizontal gene trans....

Protocol

All experiments using recombinant DNA and BSL-2 organisms were reviewed and approved by the Quinnipiac University Institutional Biosafety Committee.

1. Preparation of B. burgdorferi culture for the production of φBB-1

1. Prepare Barbour-Stoenner-Kelly medium supplemented with 6.6% heat-inactivated normal rabbit serum (BSK)¹⁵. For 1 L of 1x BSK, combine the components listed in Table 1 in 900 mL of water, adjust the pH to 7.6 using 1 N sodium hydroxide, and mix slowly at 4 °C for 2-4 h. After mixing is complete, check and readjust the pH to 7.6 if necessary....

Results

The use of bacteriophage to move DNA between more readily transformable B. burgdorferi strains or clones that are recalcitrant to electrotransformation represents another tool for the continued molecular investigation of the determinants of Lyme disease. The transduction assay described herein can be modified as needed to facilitate the movement of DNA between any clones of interest using either one or two antibiotics for the selection of potential transductants. The transduction of both prophage DNA and heterol.......

Discussion

The use of transduction could represent one method of overcoming at least some of the biological and technical barriers associated with the electrotransformation of B. burgdorferi^1,4,13,37. In many systems, bacteriophage can move host (non-prophage) DNA between bacterial cells by either generalized or specialized transduction^22,23

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 L filter units (PES, 0.22 µm pore size)	Millipore Sigma	S2GPU10RE
12 mm x 75 mm tube (dual position cap) (polypropylene)	USA Scientific	1450-0810	holds 4 mL with low void volume (for induction)
15 mL conical centrifuge tubes (polypropylene)	USA Scientific	5618-8271
1-methyl-3-nitroso-nitroguanidine (MNNG)	Millipore Sigma		CAUTION: potential carcinogen; no longer readily available, have not tested offered substitute
5.75" Pasteur Pipettes (cotton-plugged/borosilicate glass/non-sterile)	Thermo Fisher Scientific	13-678-8A	autoclave prior to use
50 mL conical centrifuge tubes (polypropylene)	USA Scientific	1500-1211
Absolute ethanol
Agarose LE	Dot Scientific inc.	AGLE-500
Bacto Neopeptone	Gibco	DF0119-17-9
Bacto TC Yeastolate	Gibco	255772
Bovine serum albumin (serum replacement grade)	Gemini Bio-Products	700-104P
Chloroform (for molecular biology)	Thermo Fisher Scientific	BP1145-1	CAUTION: volatile organic; use only in a chemical fume hood
CMRL-1066 w/o L-Glutamine (powder)	US Biological	C5900-01	cell culture grade
Erythromycin	Research Products International Corp	E57000-25.0
Gentamicin reagent solution	Gibco	15750-060
Glucose (Dextrose Anhydrous)	Thermo Fisher Scientific	BP350-500
HEPES	Thermo Fisher Scientific	BP310-500
Kanamycin sulfate	Thermo Fisher Scientific	25389-94-0
Millex-GS (0.22 µM pore size)	Millipore Sigma	SLGSM33SS	to filter sterilize antibiotics and other small volume solutions
Mitomycin C	Thermo Fisher Scientific	BP25312	CAUTION: potential carcinogen; use only in a chemical fume hood
N-acetyl-D-glucosamine	MP Biomedicals, LLC	100068
Oligonucleotides (primers for PCR)	IDT DNA
OmniPrep (total genomic extraction kit)	G Biosciences	786-136
Petri Dish (100 mm × 15 mm)	Thermo Fisher Scientific	FB0875712
Petroff-Hausser counting chamber	Hausser scientific	HS-3900
Petroff-Hausser counting chamber cover glass	Hausser scientific	HS-5051
Polyethylene glycol 8000 (PEG)	Thermo Fisher Scientific	BP233-1
Rabbit serum non-sterile trace-hemolyzed young (NRS)	Pel-Freez Biologicals	31119-3	heat inactivate as per manufacturer's instructions
Semi-micro UV transparent cuvettes	USA Scientific	9750-9150
Sodium bicarbonate	Thermo Fisher Scientific	BP328-500
Sodium chloride	Thermo Fisher Scientific	BP358-1
Sodium pyruvate	Millipore Sigma	P8674-25G
Spectronic Genesys 5	Thermo Fisher Scientific
Streptomycin sulfate solution	Millipore Sigma	S6501-50G
Trisodium citrate dihydrate	Millipore Sigma	S1804-500G	sodium citrate for BSK