Name: phage-mediated genetic manipulation of the lyme disease spirochete borrelia burgdorferi
Uploaded: 2022-09-28T14:10:00
Description: Watch this Scientific Journal Video about Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi at JoVE.com

The author wishes to thank Shawna Reed, D. Scott Samuels, and Patrick Secor for their useful discussion and Vareeon (Pam) Chonweerawong for their technical assistance. This work was supported by the Department of Biomedical Sciences and faculty research grants to Christian H. Eggers from the School of Health Sciences at Quinnipiac University.

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 &#966;BB-1
<ol>
	<li>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 th...

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. burgdorferi1,4,13,37. In many systems, bacteriophage can move host (non-prophage) DNA between bacterial cells by either generalized or specialized transduction22,23</s...

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 disease1,2,3,4. B. burgdorferi has an unusually complex genome comprised of a small linear chromosome and both linear and circular plasmids5,6. Spontaneous plasmid loss, intragenic rearrangement (movement of genes from one plasmid to another within the same organism), and horizontal gene transfer (HGT, the movement of DNA between two organisms) have given rise to a dizzying amount of genetic heterogeneity among B. burgdorferi (for an example, see Schutzer et al.7). The resulting genotypes (or &#34;strains&#34;) are all members of the same species but have genetic differences that influence their ability to transmit to and infect different mammalian hosts8,9,10,11. In this report, the term &#34;strain&#34; will be used to refer to B. burgdorferi with a particular naturally derived genetic background; the term &#34;clone&#34; will be used to refer to a strain that has been genetically modified for a particular purpose or as a result of experimental manipulation.
The molecular toolbox available for use in B. burgdorferi includes selectable markers, gene reporters, shuttle vectors, transposon mutagenesis, inducible promoters, and counter-selectable markers (for a review, see Drektrah and Samuels12). The effective use of these methodologies requires the artificial introduction of heterologous (foreign) DNA into a B. burgdorferi strain of interest. In B. burgdorferi, the introduction of heterologous DNA is achieved almost exclusively via electroporation, a method that utilizes a pulse of electricity to make a bacterial membrane transiently permeable to small pieces of DNA introduced into the media1. The majority of the cells (estimated to be &#8805;99.5%) are killed by the pulse, but the remaining cells have a high frequency of retaining the heterologous DNA13. Although considered to be among the most highly efficient methods of introducing DNA into bacteria, the frequency of electroporation into B. burgdorferi is very low (ranging from 1 transformant in 5 &#215; 104 to 5 &#215; 106 cells)13. The barriers to achieving higher frequencies of transformation seem to be both technical and biological. Technical barriers to the successful electroporation of B. burgdorferi include both the amount of DNA (&#62;10 &#956;g) that is necessary and the requirement of the spirochetes to be in exactly the correct growth phase (mid-log, between 2 &#215; 107 cells&#183;mL&#8722;1 and 7 &#215; 107 cells&#183;mL&#8722;1) when preparing electrocompetent cells12,13. These technical barriers, however, may be easier to overcome than the biological barriers.
Lyme disease researchers recognize that B. burgdorferi clones can be divided into two broad categories with respect to their ability to be manipulated genetically13,14. High passage, lab-adapted isolates are often readily transformed but usually have lost the plasmids essential for infectivity, behave in a physiologically aberrant fashion, and are not able to infect a mammalian host or persist within a tick vector12,13. While these clones have been useful for dissecting the molecular biology of the spirochete within the lab, they are of little value for studying the spirochete within the biological context of the enzootic cycle. Low-passage infectious isolates, on the other hand, behave in a physiologic manner reflective of an infectious state and can complete the infectious cycle but usually are recalcitrant to the introduction of heterologous DNA and are, therefore, difficult to manipulate for study12,13. The difficulty in transforming low-passage isolates is related to at least two different factors: (i) low-passage isolates often tightly clump together, particularly under the high-density conditions required for electroporation, thus blocking many cells from either the full application of the electrical charge or access to the DNA in the media13,15; and (ii) B. burgdorferi encodes at least two different plasmid-borne restriction-modification (R-M) systems that may be lost in high-passage isolates14,16. R-M systems have evolved to allow bacteria to recognize and eliminate foreign DNA17. Indeed, several studies in B. burgdorferi have demonstrated that transformation efficiencies increase when the source of the DNA is B. burgdorferi rather than Escherichia coli13,16. Unfortunately, acquiring the requisite high concentration of DNA for electroporation from B. burgdorferi is an expensive and time-consuming prospect. Another potential concern when electroporating and selecting low-passage isolates is that the process seems to favor transformants that have lost the critical virulence-associated plasmid, lp2514,18,19; thus, the very act of genetically manipulating low-passage B. burgdorferi isolates via electroporation may select for clones that are not suitable for biologically relevant analysis within the enzootic cycle20. Given these issues, a system in which heterologous DNA could be electrotransformed into high-passage B. burgdorferi clones and then transferred into low-passage infectious isolates by a method other than electroporation could be a welcome addition to the growing collection of molecular tools available for use in the Lyme disease spirochete.
In addition to transformation (the uptake of naked DNA), there are two other mechanisms by which bacteria regularly take up heterologous DNA: conjugation, which is the exchange of DNA between bacteria in direct physical contact with each other, and transduction, which is the exchange of DNA mediated by a bacteriophage21. Indeed, the ability of bacteriophage to mediate HGT has been used as an experimental tool for dissecting the molecular processes within a number of bacterial systems22,23,24. B. burgdorferi is not naturally competent for the uptake of naked DNA, and there is little evidence that B. burgdorferi encodes the apparatus necessary to promote successful conjugation. Previous reports have described, however, the identification and preliminary characterization of &#966;BB-1, a temperate bacteriophage of B. burgdorferi25,26,27,28. &#966;BB-1 packages a family of 30 kb plasmids found within B. burgdorferi25; the members of this family have been designated cp32s. Consistent with a role for &#966;BB-1 in participating in HGT among B. burgdorferi strains, Stevenson et al. reported an identical cp32 found in two strains with otherwise disparate cp32s, suggesting a recent sharing of this cp32 between these two strains, likely via transduction29. There also is evidence of significant recombination via HGT among the cp32s in an otherwise relatively stable genome30,31,32,33. Finally, the ability of &#966;BB-1 to transduce both cp32s and heterologous shuttle vector DNA between cells of the same strain and between cells of two different strains has been demonstrated previously27,28. Given these findings, &#966;BB-1 has been proposed as another tool to be developed for the dissection of the molecular biology of B. burgdorferi.
The goal of this report is to detail a method for inducing and purifying phage &#966;BB-1 from B. burgdorferi, as well as provide a protocol for performing a transduction assay between B. burgdorferi clones and selecting and screening potential transductants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L filter units (PES, 0.22 &#181;m pore size)</td>
			<td>Millipore Sigma</td>
			<td>S2GPU10RE</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm tube (dual position cap) (polypropylene)</td>
			<td>USA Scientific</td>
			<td>1450-0810</td>
			<td>holds 4 mL with low void volume (for induction)</td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes (polypropylene)</td>
			<td>USA Scientific</td>
			<td>5618-8271</td>
			<td></td>
		</tr>
		<tr>
			<td>1-methyl-3-nitroso-nitroguanidine (MNNG)</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td>CAUTION: potential carcinogen; no longer readily available, have not tested offered substitute</td>
		</tr>
		<tr>
			<td>5.75&#34; Pasteur Pipettes (cotton-plugged/borosilicate glass/non-sterile)</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-678-8A</td>
			<td>autoclave prior to use</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes (polypropylene)</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>Dot Scientific inc.</td>
			<td>AGLE-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Neopeptone</td>
			<td>Gibco</td>
			<td>DF0119-17-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto TC Yeastolate</td>
			<td>Gibco</td>
			<td>255772</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (serum replacement grade)</td>
			<td>Gemini Bio-Products</td>
			<td>700-104P</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (for molecular biology)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1145-1</td>
			<td>CAUTION: volatile organic; use only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>CMRL-1066 w/o L-Glutamine (powder)</td>
			<td>US Biological</td>
			<td>C5900-01</td>
			<td>cell culture grade</td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Research Products International Corp</td>
			<td>E57000-25.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin reagent solution</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (Dextrose Anhydrous)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP350-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>25389-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GS (0.22 &#181;M pore size)</td>
			<td>Millipore Sigma</td>
			<td>SLGSM33SS</td>
			<td>to filter sterilize antibiotics and other small volume solutions</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP25312</td>
			<td>CAUTION: potential carcinogen; use only in a chemical fume hood</td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>MP Biomedicals, LLC</td>
			<td>100068</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides (primers for PCR)</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPrep (total genomic extraction kit)</td>
			<td>G Biosciences</td>
			<td>786-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (100 mm &#215; 15 mm)</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroff-Hausser counting chamber</td>
			<td>Hausser scientific</td>
			<td>HS-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroff-Hausser counting chamber cover glass</td>
			<td>Hausser scientific</td>
			<td>HS-5051</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000 (PEG)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP233-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit serum non-sterile trace-hemolyzed young (NRS)</td>
			<td>Pel-Freez Biologicals</td>
			<td>31119-3</td>
			<td>heat inactivate as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Semi-micro UV transparent cuvettes</td>
			<td>USA Scientific</td>
			<td>9750-9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Millipore Sigma</td>
			<td>P8674-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectronic Genesys 5</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate solution</td>
			<td>Millipore Sigma</td>
			<td>S6501-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Millipore Sigma</td>
			<td>S1804-500G</td>
			<td>sodium citrate for BSK</td>
		</tr>
	</tbody>
</table>

phage-mediated genetic manipulation of the lyme disease spirochete borrelia burgdorferi

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 &#966;BB-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 &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

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...

Watch this Scientific Journal Video about Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi at JoVE.com

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

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 &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

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

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

This protocol is significant because it describes another important tool for dissecting the molecular biology of the Lyme disease agent Borrelia burgdorferi. The main advantage of this technique is that by using phage transduction, it provides an alternative method for introducing DNA in the Borrelia burgdorferi without requiring the application of an electric pulse, as in electroporation. This method could be used to provide further insight into the molecular mechanisms used by Borrelia burgdorferi to persist in its enzootic cycle and ultimately cause Lyme disease in humans.To begin, inoculate 150 microliters of the appropriate Borrelia burgdorferi clone into 15 milliliters of BSK in tightly-capped sterile conical centrifuge tubes for the transduction protocol. Then, supplement the medium with the appropriate concentration of antibiotics or a combination of antibiotics for the selection and maintenance of heterologous DNA within the Borrelia burgdorferi clone and incubate the sample at 33 degrees Celsius. After the culture has grown up, centrifuge the appropriate volume of culture at 6, 000 G for 10 minutes.After decanting the supernatant, resuspend the pellet in four milliliters at fresh BSK and transfer the sample to the smallest sterile tube available to hold the sample with minimal head space. Then, add the appropriate amount of inducing agent to the recommended concentration based on a culture volume of four milliliters to induce phage production, cap the tube tightly, and mix thoroughly. Incubate the sample at 33 degrees Celsius for two to four hours.Then, transfer the sample to a 15-milliliter centrifuge tube. Centrifuge the sample at 6, 000 G for 10 minutes. After decanting the supernatant, resuspend the cell pellet in 15 milliliters of BSK.Supplement the prepared culture with the appropriate antibiotic concentration described in the manuscript, while incubating the sample at 33 degrees Celsius for 72 to 96 hours. To prepare solutions for PEG precipitation, prepare 500 milliliters of five-molar sodium chloride, 500 milliliters of 40%PEG, and 100 milliliters of suspension medium as described in the manuscript. To sterilize, autoclave the solution, cool prior to use, and store room temperature or four degrees Celsius.For PEG precipitation of phage from the donor Borrelia burgdorferi clone, after 72 to 96 hours of incubation, centrifuge the samples at 8, 000 G for 20 minutes at four degrees Celsius. Decant the supernatant into a clean, 50-milliliter conical tube and dispose of the cell pellet. Add five-molar sodium chloride to a final concentration of one molar.After mixing well, rock gently at room temperature for one hour. Centrifuge the samples at 8, 000 G for 10 minutes at four degrees Celsius. After decanting the supernatant into a clean 50-milliliter conical tube as demonstrated previously, add 40%PEG-8000 solution to the supernatant to a final concentration of 10%Mix well and set on ice for more than one hour, up to overnight.Centrifuge to the samples at 8, 000 G for 20 minutes at four degrees Celsius as demonstrated previously. Discard the supernatant and remove as much excess liquid as possible without losing any pellet, which contains the phage particles. Resuspend the pellet in a minimal volume of suspension medium using the suspension medium to wash down the side of the bottle and collect any potential phage particles.Treat the recovered phage sample with an equal volume of chloroform based on the volume of resuspension. Mix the sample well and centrifuge at 8, 000 G for 10 minutes. Then, remove the aqueous layer to a clean tube, avoiding any of the thick interface layers.After determining the volume recovered, following the first chloroform treatment, again treat the sample with an amount of chloroform equal to 10%of that volume. Transfer the aqueous layer to a clean tube while being careful to avoid any of the interface or organic layers. Use the phage immediately or store it at four degrees Celsius.After preparing Borrelia burgdorferi cultures to be used as the recipient in transduction assays as demonstrated previously, centrifuge the volume of culture at 6, 000 G for 10 minutes. Decant the supernatant and resuspend the pellet in 14.5 milliliters of fresh BSK. Add less than or equal to 500 microliters of PEG-precipitated phage sample to the culture of the recipient clone.After mixing well, incubate at 33 degrees Celsius for 72 to 96 hours. Perform the selection of transductants by solid-phase plating after mixing with PEG-precipitated phage as described in the manuscript. Depending on the background of the recipient clone, check that the colonies appear within the agarose on the selection plates after 10 to 21 days of incubation.Pick at least 5 to 10 colonies that grow on the plate in the presence of both antibiotics using sterilized cotton-plugged 5.75-inches borosilicate pipette and inoculate them into 1.5 milliliters of BSK with the appropriate antibiotic. The PCR amplification of the genes was performed on 10 of the clones encoding kanamycin and gentamycin resistance. The kanamycin resistance gene could be amplified from the donor c1673 and the potential transductants but not the recipient c1706.Similarly, the gentamycin resistance gene could be amplified from the recipient c1706 and the potential transductants but not the donor, representing transduction events. To demonstrate that the kanamycin resistance cassette was transduced by 5BB1 from the donor into the recipient. The background of the transductants was determined using strain-specific markers.The c1673 clone and the CA-112A background encodes specific amplicons four, five, and six, whereas c1706, which has a high-passage B31 background, does not. Similarly, the transductants one and two were missing amplicons four, five and six, demonstrating that the clones have the c1706 background and have acquired the kanamycin resistance gene from c1673. For the PEG precipitation of phage, perform all steps carefully to maximize the yield of phage and treat the resuspended phage thoroughly with chloroform to remove as much PEG and culture media contaminants as possible prior to phage use and storage.Once the transduction assay has been successfully performed and the transductant verified, these clones are then available to answer the questions that require genetically-modified Borrelia burgdorferi. The development of this technique will hopefully allow researchers to genetically manipulate Borrelia burgdorferi strains for which the introduction of DNA via electroporation is currently difficult.

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