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[25] G e n e t i c S y s t e m s for M y c o b a c t e r i a
By WILLIAM R. JACOBS, JR., GANJAMV. KALPANA, JEFFREY D. CIRILLO, LISA PASCOPELLA, SCOTT B. SNAPPER, RUPA A. UDANI, WILBUR JONES, RAI3L G. BARLETTA, a n d BARRY R. BLOOM
Introduction The ability to perform genetic analyses on bacteria has provided powerful tools and experimental systems to unravel fundamental biological processes. The advances of recombinant DNA technologies have ignited the development of genetic systems for bacteria that are difficult to work with. Clearly, the genus Mycobacterium contains a set of the most difficult bacterial species to manipulate experimentally. Despite its discovery over 100 years ago, the leprosy bacillus, Mycobacterium leprae, remains unable to be cultivated in the laboratory except in mouse footpads or in the ninebanded armadillo. Although Robert Koch pioneered the work of pure culture with Mycobacterium tuberculosis, the ability to extend genetic analyses beyond these initial studies has been greatly hampered by the slow growth of mycobacteria and the stubborn tendency to clump. The tuberculosis vaccine strain, BCG (bacille Calmette Gu6rin) has been used to vaccinate more individuals than any other live bacterial vaccine, yet little is known about mycobacterial gene structure and expression. The recent development of phage,l'2 plasmid,2 and gene replacement3 systems for the introduction of recombinant DNA into mycobacteria has opened up a new era of research on members of the genus Mycobacterium. Indeed, the use of these technologies should pave the way for the elucidation of mycobacterial virulence determinants and a detailed understanding of the mechanisms of drug action and resistance, as well as the development of recombinant multivalent BCG vaccines 1'4 capable of engendering immune responses to a variety of viral, bacterial, or parasitic antigens. The goal of this chapter is to bring together an array of recently developed methods and techniques which enable researchers to genetically manipulate mycobacterial species. I W. R. Jacobs, Jr., M. Tuckman, and B. R. Bloom, Nature (London) 327, 532 (1987). 2 S. B. Snapper, L. Lugosi, A. Jekkel, R. Melton, T. Kieser, B. R. Bloom, and W. R. Jacobs, Jr., Proc. Natl. Acad. Sci. U.S.A. 8S, 6987 (1988). 3 R. N. Husson, B. E. James, and R. A. Young, J. Bacteriol. 172, 519 (1990). 4 B. R. Bloom, J. Immunol. 10, i (1986).
METHODS IN ENZYMOLOGY, VOL. 204
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Mycobacterial Strains The mycobacteria fall into two general categories based on their growth rates. The fast-growing mycobacteria, consisting of such strains as M. smegmatis, M. phlei, M. aurum, and M. fortuitum, have doubling times of 2-3 hr and will yield a colony from a single cell in 3-4 days. In contrast, the slow-growing mycobacteria such as M. tuberculosis, BCG, or M. avium strains double every 18-26 hr and thus yield colonies from single cells in 14-28 days. Although it is likely that the vectors and methodologies described here are applicable to many mycobacterial species, we have focused our efforts on the fast-growing M. smegmatis and the slow-growing BCG. The strains used are (I) M. smegmatis strain mcZ6, a single colony isolate of the predominant colony type found in the ATCC 607 culture; (2) M. smegmatis strain mc2155, an efficient transformation mutant 5 of mc26; and (3) BCG-Pasteur obtained as a lyophilized pellet from the Statens Serum Institute in Copenhagen, Denmark.
Biohazard Considerations Biosafety level 3 (BL3) containment facilities and corresponding practices and equipment are appropriate for activities involving the propagation and manipulation of M. tuberculosis and M. boris; all other mycobacterial species including BCG and M. avium can be safely handled in BL2 containment facilities. 6 Experiments involving the introduction of recombinant mycobacterial DNA into Escherichia coli or any mycobacterial host require that the investigator obtain approval from the Institution's Biosafety Committee before the initiation of experiments. 7 The National Institutes of Health Recombinant DNA Advisory Committee (RAC) recommends that experiments involving the introduction of recombinant DNA from a class 3 agent, such as M. tuberculosis, into a class 2 agent such as M. smegmatis be performed using BL3 containment facilities. The deliberate transfer of a gene conferring drug resistance whose acquisition could compromise the use of a drug to control the pathogen, such as the introduction of isoniazid resistance into M. tuberculosis, can only be
5 S. B. Snapper, R. E. Melton, S. Mustafa, T. Kieser, and W. R. Jacobs, Jr., Mol. Microbiol. 4, 1123 (1990). 6 U.S. Dept. of Health and Human Services, CDC/NIH. "Biosafety in Microbiological and Biomedical Laboratories." U.S. Government Printing Office, Washington, D.C. HHS Publication No. (CDC) 86-8395 (1986). 7 U.S. Department of Health and Human Services, Federal Register 51, 16957 (1986).
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performed following approval from both the RAC and the Institutional Biosafety Committee.
Growth and Maintenance of Mycobacterial Strains
Media For Growth of Mycobacteria in Liquid Culture M - A D C - T W broth: Dissolve 4.7 g of Difco (Detroit, MI) Middlebrook 7H9 broth base and 2 ml glycerol in 900 ml deionized water. Autoclave 20 min. After cooling, add 100 ml albumin-dextrose complex (ADC) enrichment and 2.5 ml of 20% Tween 80 solution. The albumin component of the ADC enrichment is essential for the growth of BCG but is not needed for M. smegmatis growth. ADC enrichment: Dissolve 2 g glucose, 5 g bovine serum albumin fraction V, (Boehringer Mannheim, Indianapolis, IN), and 0.85 g NaC1 in 100 ml deionized water. Filter sterilize and store at 4 °. 20% Tween 80: Add 20 ml of polyoxyethylene sorbitan monooleate to 80 ml deionized water, heat at 55 ° for 30 min to dissolve completely, and filter sterilize.
For Colony Titrations, Transformations, and Transductions with Shuttle Phasmids Middlebrook 7H10 agar: Add 19 g Middlebrook 7H10 agar to 900 ml deionized water, autoclave 20 rain, and allow to temper to 55 °. Add 100 ml ADC enrichment and pour 40-45 ml per plate. Cycloheximide can be added at a final concentration of 50 t~g/ml to inhibit mold contamination. Kanamycin is added to a final concentration of 10 t~g/ml for the selection of shuttle phasmid transductants or transformants. For Propagating Mycobacteriophage and Shuttle Phasmids. It is most important not to have Tween 80 in media used to propagate phage. DB+ bottom agar: Add 20 g Dubos oleic acid agar base, 1 g NaCI, and 7.5 g glucose to 1 liter deionized water. Autoclave 20 rain, temper to 55 °, and add MgSO4 and CaCI2 to final concentrations of 10 and 2 raM, respectively. Mycobacteriophage top agar: Add 0.5 g Middlebrook 7H9 broth base, 0.1 g NaCI, 0.75 g glucose, and 0.7 g agar to 100 ml deionized water. For BCG, supplement this by adding 1.0 g proteose peptone #3
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(Difco), 0.5 g yeast extract (Difco), and 5 ml of a 20% glycerol solution (v/v).
Growth of Mycobacterium smegmatis and BCG The slow growth of the mycobacteria necessitates that excellent aseptic technique be followed since most contaminants will outgrow mycobacteria, particularly the slow-growing mycobacteria. Since the time required for a single cell to yield a colony might be up to 3 to 6 weeks, which is often essential for genetic analyses, agar plates onto which bacteria cells are plated must be poured thick and wrapped with Parafilm both to prevent desiccation of the media and to reduce possible risks of mold contamination. Tween 80 is an essential component of mycobacterial media that reduces the clumping of mycobacteria and aids in the preparation of single cell suspensions. However, microscopic analyses will usually demonstrate clumps of mycobacterial cells even when the cultures are grown in Tween 80. Mycobacterium smegmatis will clump less than most of the other fastgrowing mycobacteria and can yield a reasonably satisfactory single cell suspension when grown in M-ADC-TW broth, in baffled flasks, with shaking at 37°. In contrast, BCG will clump considerably if grown like M. smegmatis. We have found it best either to grow BCG standing, for phage work, or to grow it in tissue culture roller bottle flasks to yield high levels of reasonably unclumped viable cells. Mycobacteriophage and Shuttle Phasmids Historically, mycobacteriophage have been used to type various mycobacterial isolates. Novel phage vectors, termed shuttle phasmids, have been constructed from the mycobacteriophage TM4 and L1.1'2 Shuttle phasmids s can replicate in mycobacteria as phage capable of undergoing lysis or lysogeny. These vectors can also replicate in E. coli as cosmids and, thus, be packaged into bacteriophage h heads to facilitate subsequent cloning of additional genes. 8
Preparation of High-Titer Lysates of Mycobacteriophage and Shuttle Phasmids 1. Prepare M. smegmatis mc26 cells for a phage infection by inoculating 0.1 ml of the starter culture into 25 ml of M-ADC-TW broth in a 250-ml baffled flask. Shake the flask at 100 rpm on a platform 8 W. R. Jacobs, Jr., S. B. Snapper, M. Tuckman, and B. R. Bloom, Rev. Infect. Dis. 11, $404 (1989).
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shaker at 37° overnight. Although mycobacteriophage infections are inhibited by Tween 80, overnight cultures of M. smegmatis contain Tween hydrolase that destroys most of the added Tween. However, the addition of Tween 80 is still necessary to yield an unclumped lawn of mycobacterial cells. We have found it best for BCG phage lawns to use 10-day-old standing cultures of BCG grown at 37° from a 1 : 1000 dilution of an exponential BCG culture. 2. Dilute the phage or shuttle phasmid, 10-fold serially, in MP buffer (10 mM Tris, pH 7.6, 100 mM NaC1, 10 mM MgSO 4 , 2 mM CaCI 2) to obtain approximately 5 × 104 to 105 plaque forming units (pfu) per milliliter. 3. Mix 2.0 ml of mc26 cells ( - 3 x 108 cells/ml) with 1.0 ml of the phage or shuttle phasmid diluted to 5 x 104 pfu/ml in a sterile 13 x 100 mm culture tube. Incubate at 37° for 30 min to allow adsorption to Occur.
4. Pipette 0.3 ml of the phage-cell mixture to a culture tube containing 3.0 ml of DB~b top agar from M. smegmatis or DB~b supplemented top agar for BCG, mix the phage-cell suspension with the top agar by gently rotating the tube in the palms of your hands, and aseptically pour on top of a DBth plate. Allow the agar to solidify. Invert the plate and incubate at 37° for 24-36 hr for M. smegmatis or 7-10 days for BCG. For each lysate, we normally prepare five plates. 5. Pipette 5 ml of MP buffer on each plate. Set plates upright at 4 ° overnight. 6. Pipette off as much liquid as possible (usually - 3 ml) from each plate lysate and combine all of the lysate fluids in a 50-ml polypropylene tube. Centrifuge the lysates at 4° at 3000 g for 10 rain. Filter the supernatant fluid through a 0.45-/zm filter unit. Transfer the filtered phage lysate preparation to a sterile phage bottle. Store at 4 °. Note: Do not add chloroform to mycobacteriophage lysates as most mycobacteriophage contain lipids and are inactivated by nonpolar solvents.
Phage Purification and Isolation of DNA Mycobacteriophage can be easily purified and concentrated on CsCl density gradients as described for E. coli phage.9 The procedures described here have given high yields of phage particles from mycobacteriophage D29, L1, TM4, and shuttle phasmid derivatives. DNA can be efficiently 9 R. W. Davis, D. Botstein, and J. R. Roth, "Advanced Bacterial Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
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isolated from the purified phage particles following proteinase K treatment, phenol-chloroform extractions, and ethanol precipitations.
Transfection of Phage DNA by Electroporation 1. Transfect phage DNA by electroporation. A detailed protocol for electroporation of mycobacteria is described below. Basically, the same procedure is carried out for transfection with phage DNA. The following considerations should be taken into account for transfection protocols: to maximize the yield of plaques, it is essential to dilute the cell suspension after electroporation, and incubate further for I hr at 37°. The dilution is important to reduce the level of harmful chemicals (radicals) generated during electroporation, and the subsequent incubation allows the mycobacterial cells to recover from the electric shock. 2. Mix 0.1 ml of an appropriate dilution of the electroporation mixture with 0.2 ml of a freshly grown culture (logarithmic to early stationary culture) of mycobacterial cells. Then add 3 ml of soft agar and plate. Usually, transfection frequencies around 105 pfu//zg of phage DNA are obtained.
Construction of Shuttle Phasmids Shuttle phasmids are chimeric constructs between a mycobacteriophage and an E. coli plasmid, in which the plasmid is inserted in a nonessential region of the phage genome. Usually, an E. coli cosmid is used as a plasmid, since the possibility of performing in vitro packaging greatly facilitates the construction of recombinant structures. The steps involved in making such constructs are as follows: 1. Ligate mycobacteriophage DNA at high DNA concentrations (-> 100 /zg/ml, usually between 250 and 500/xg/ml). 2. Digest the ligated mycobacteriophage DNA partially with a frequent cutter (e.g., Sau3AI), so that the majority of the fragments obtained are in the range of 44 kilobases (kb) (if the cosmid used is - 6 kb, like pHC79). At this stage it is possible to fractionate the 44-kb fragments if desired. 3. Linearize the cosmid vector DNA with a compatible restriction enzyme (usually the corresponding hexamer, e.g., BamHI); treat with alkaline phosphatase if desired. 4. Ligate the linearized cosmid vector with the digested mycobacteriophage DNA from Step 2 at high DNA concentrations (250-500 /zg/ml) so that concatemeric DNA is obtained, with intermingled fragments of mycobacteriophage and cosmid DNA. 5. In vitro package, using commercially available packaging mixes.
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The ligated DNA is packaged in h particles by virtue of the cos sites present in the cosmid vector. At this step a transducing phage lysate containing the recombinant DNA is obtained. With the phage lysate transduce an appropriate E. coli host containing mutations in the hsdR, mcrA, and mcrB genes, such as ER1451 (New England Biolabs, Beverly, MA). Select transductants containing recombinant cosmid DNA by plating on appropriate antibiotic plates (L agar plus 25/zg/ml of ampicillin for pHC79). Pool transductants and isolate plasmid DNA by standard procedures. The plasmids isolated from this pool represent an insertion library of the cosmid vector in the mycobacteriophage genome. Only those plasmids with insertions within a dispensable region of the mycobacteriophage genome will give rise to shuttle phasmids. Electroporate an appropriate mycobacterial host (mc26 or mc2155 for L1 and TM4), using the pool of recombinant plasmids, as previously described. Screen the mycobacteriophage plaques obtained using the cosmid vector as probe. Positive hybridizing plaques represent true shuttle phasmids. At this stage, we have observed with the construction of TM4-derived shuttle phasmids that almost all the resulting plaques have lost the insertion of the cosmid DNA (1 positive for every 400 plaques), presumably the result of a recombination event between multiple recombinant cosmid::phage molecules entering a cell and recombining. Shuttle phasmids can be isolated, ligated, in vitro packaged into bacteriophage h heads, and transduced into E. coli cells where they replicate as cosmids conferring antibiotic resistance.
Cloning Genes in Shuttle Phasmids
Unique cloning sites within the cosmid vector can be useful for cloning foreign DNA, provided that they are absent from the phage genome. For example, both TM4 and L1 lack EcoRI and HindlII sites within the phage genome, and therefore the unique EcoRI site or HindlII sites in pHC79 can be used to introduce additional foreign DNA. The size of foreign DNA that can be cloned in a shuttle phasmid is constrained by the size requirements of the mycobacteriophage packaging machinery, as well as by the cosmid packaging mechanism. In this way, additional genes, 2 to 4 kb in size, can be efficiently cloned into shuttle phasmids. The procedure can be summarized as follows: 1. Ligate shuttle phasmid DNA so that long concatemers are obtained. 2. Digest shuttle phasmid DNA with an appropriate restriction enzyme (e.g., EcoRI for L1 shuttle phasmids) to yield large DNA vector
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molecules that can be ligated to a gene of choice with compatible restriction enzyme ends. . After ligation, in vitro package, and proceed as described above for the construction of shuttle phasmids. By using the temperate shuttle phasmid phAE15, 2 derived from mycobacteriophage LI, one can select or screen for mycobacterial colonies carrying the recombinant DNA with the gene of interest.
E l e c t r o p o r a t i o n o f Mycobacterium smegmatis a n d B C G
The highest efficiencies for transformation of mycobacteria have been obtained using electroporation. We have found that mycobacteria can withstand very high voltages for extended periods of time. Thus, a highresistance electroporation buffer such as 10% glycerol is used along with a high parallel resistance. This method results in long time constants, which have been shown to give the highest efficiencies of transformation. The protocol which we use is similar to that used for E. coli.10 1. Inoculate 1 liter of M - A D C - T W broth with 10 ml of a 10-to 15-day culture of BCG (A600 = 0.5-1.0). In the case ofM. smegmatis, 1 liter of M - A D C - T W broth can be directly inoculated from a frozen stock or colony directly. 2. Incubate at 37 ° until the A600reaches 0.5-1.0. For BCG this period varies greatly, from 7 to 25 days, depending on the passage of the culture and growth conditions; M. smegmatis, however, is almost always at the correct stage of growth by 48 hr. 3. Harvest in 250-ml centrifuge bottles. Centrifuge for I0 min at 10,000 rpm and 4 °. Discard supernatant, 4. Resuspend each BCG pellet in 5 ml of ice-cold 10% glycerol, pool them into two 15-ml polypropylene conical tubes, and centrifuge for 10 min at 3000 rpm and 4 °. Mycobacterium smegmatis, however, should be directly suspended in the centrifuge bottles in 250 ml of 10% glycerol and centrifuged as before. 5. Resuspend the BCG pellets in I0 ml cold 10% glycerol. At this point, M. smegmatis pellets may be resuspended in 10 ml cold 10% glycerol, pooled into two 50-ml polypropylene conical tubes, and the volume raised to 50 ml with 10% glycerol. Centrifuge for I0 min at 3000 rpm and 4°. 6. Repeat the wash in Step 5. 7. Resuspend each pellet to a final volume of 1 ml in 10% glycerol. l0 W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988).
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9.
10.
1 I. 12.
13.
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The protocol can be continued with the freshly prepared cells as shown below, or, at this step, the cells can be aliquoted into Eppendorf tubes and frozen quickly in dry ice-ethanol to be used later by thawing slowly at room temperature (sometimes the thawed cells will contain salts and cause arcing; this can be prevented by washing the cells in the Eppendorf tubes with 10% glycerol once or twice). Place DNA (5 pg-5/zg; 5 gl volume, maximum) and 50 tzl of cells in an Eppendorf tube and mix well by pipetting up and down. Place on ice 1 min. Set the voltage of the pulser (Bio-Rad, Richmond, CA) to 2500 V, 25/zF, and the pulse controller (Bio-Rad) to 1000 ohms. Sometimes the cells will arc with a high parallel resistance; washing the cells further with 10% glycerol will usually prevent this. If DNA containing high salt is added, either a lower resistance in the pulse controller must be used or the DNA must be ethanol precipitated and washed with 70% (v/v) ethanol at least once. If a lower resistance is used, lower transformation frequencies will result; therefore, ethanol precipitation is recommended. Transfer the solution containing the cells and DNA into a cuvette with a 0.2-cm electrode gap (Bio-Rad). Tap the cuvette against the bench several times to get the cells to the bottom of the cuvette and to remove as many bubbles as possible. Place the cuvette in the pulser and expose to one pulse (time constants are usually between 15.0 and 25.0 msec). For BCG electroporations add 0.4 ml of M - A D C - T W medium directly to the cuvette and suspend the cells well in it. For M. smegmatis add 1 ml of M - A D C - T W broth, suspend the cells in it, and transfer to a round-bottomed 15-ml polypropylene tube. Incubate at 37° approximately 3 hr to allow antibiotic expression. Plate the cells on selective medium.
Both the M. smegmatis and BCG electroporation procedures are extremely sensitive to the concentration of salts present in the DNA sample used. The DNA used should be washed with 70% ethanol if there is any possibility of the presence of excess salt. Also, it is important to add as small a volume of DNA as possible to prevent dilution of the bacterial cell concentration in the cuvette. Ligations must be ethanol precipitated and washed with 70% ethanol before they can be used (it is possible to use small amounts of a ligation, but significantly lower transformation efficiencies will result). After the last wash, if the packed cell volume is not very close to or is more than 1 ml, do not raise the volume to 1 ml final,
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just add enough 10% glycerol to allow the cells to be pipetted easily. Fresh cells in our hands give transformation efficiencies above 106 transformants/ /zg (using a 10-kb plasmid), and frozen cells give efficiencies which vary from 104 to 106 transformants//zg. Chromosomal DNA Isolation and Plasmid Isolation from Mycobacterial Cells
Isolation of High Molecular Weight Mycobacterial Chromosomal DNA Using Mini-Beadbeater Cell Disruptor 1. Grow 100 ml of culture ofM. smegmatis or BCG cells to approximately 3 x 108 cells/ml. Normally, we use overnight M. smegmatis cultures started from a 0.1-ml starter culture or 7- to 9-day-old BCG cultures that have been grown with shaking. Large numbers of freshly grown ceils are critical to obtaining good yields of high molecular weight DNA. 2. Pellet the cells by centrifuging in 50-ml polypropylene tubes in a Sorvall RT6000 centrifuge at 3000 rpm for 10 min at 4°. 3. Carefully, pour off the supernatant fluid and resuspend the pellet in 0.5 ml of homogenization buffer (0.3 M Tris, pH 8, 0.1 M NaCI-6 m M EDTA). 4. Transfer the resuspended cells to conical 2-ml screw-cap vial which is one-fourth to one-half filled with 0.5-mm acid-washed sterile glass beads. 5. Place the vial on the Mini-Beadbeater cell disruptor (Biospec Products, BartlesviUe, OK) and beat it for 1 rain. 6. Transfer the entire contents of the tube to a 15-ml conical polypropylene tube. Wash the 2-ml Beadbeater vial with 1 ml of homogenization buffer and add this to the disrupted cell suspension. 7. Extract with an equal volume of phenol-chloroform (1 : 1) solution. Use Tris-buffered neutralized phenol. Repeat the extraction one more time. After each extraction, spin the tubes in a Sorvall RT6000 (Dupont, Wilmington, DE) centrifuge at 3000 rpm for 10 min. 8. Peform one chloroform-isoamyl (24-1) extraction and then ethanol precipitated with 1/10 volume of 3 M sodium acetate, pH 5.0, and 2 volumes of cold ethanol. 9. Place the tubes at - 7 0 ° for 30 min or - 2 0 ° overnight for ethanol precipitation. 10. Spin the tubes for 10-15 min at 3000 rpm in Sorvall RT6000 centrifuge.
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11. Pour off the ethanol and wash the pellet with 2 ml of cold 70% ethanol. 12. Spin the precipitate again for 5 min in a SorvaU RT6000 centrifuge at 3000 rpm. 13. Pour off the ethanol and dry the pellet. 14. Resuspend the pellet in I ml of TE (10 mM Tris-1 mM EDTA) buffer. Do not shake or vortex vigorously. Let the tube sit for a few minutes and then just gently mix up and down 2-3 times. If the pellet still does not dissolve, add more TE. 15. Digest RNA by adding RNase A at I00 /zg/ml using 10 mg/ml boiled stock. Incubate at 37° for 30 min. 16. Extract with an equal volume of phenol-chloroform twice as before. 17. Extract with an equal volume of chloroform-isoamyl alcohol once. 18. Ethanol precipitate with 1/I0 volume of 3 M sodium acetate and 2 volumes of ethanol. 19. Pellet the DNA, pour offthe supernatant fluid, dry, and resuspend the pellet in 1 ml TE.
Plasmid DNA Isolation from Mycobacterial Cells We have adapted the alkaline lysis protocol 1~with the following modifications: 1. Grow 5 to 10 ml of the mycobacterial cell culture to approximately saturation (overnight for the fast growers, several days for slow growers). 2. Harvest the cells by centrifugation, resuspend the pellet in 150/xl of GTE (25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM glucose) containing 10 mg/ml of lysozyme, and incubate at 37° overnight. 3. Add 2 volumes (300/zl) of fresh 0.2 M N a O H - I % sodium dodecyl sulfate (SDS) and mix by inversion. Incubate at 4 ° for 10 min. 4. Add 1.5 volumes (225 /zl) of 5 M potassium acetate and mix by inversion. Incubate at 4 ° for 10 min. 5. Centrifuge in microcentrifuge for 30 min to 1 hr. This step is critical to obtain a good plasmid preparation. 6. Remove and extract the supernatant with an equal volume of CPI (chloroform-phenol-isoamyl alcohol, 24 : 25 : 1). 7. Transfer the aqueous phase to a clean tube and add 2 volumes (1200/zl) of room temperature ethanol, incubate for 5 min at room temperature, spin down the DNA pellet, wash with 70% ethanol, and dry briefly under reduced pressure. u H. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979).
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8. Resuspend in 25 to 50/zl of TE buffer, then incubate at 65° for 15 min. Insertional Mutagenesis A method of generating random chromosomal mutations by inserting a piece of DNA containing a selectable marker gene is essential for the mycobacteria since it permits one to select mutants arising from a single mutagenic event directly, even from a pool of clumped cells. Insertional mutagenesis using transposons has been a valuable method for mutational analysis. In the absence of a mycobacterial transposon that transposes at high frequency we have developed an indirect method of random mutagenesis, a random shuttle mutagenesis. 12 This method was developed by extending the strategy of targeted mutagenesis (Fig. 1). Shuttle mutagenesis of a targeted gene involves three steps: cloning of the desired gene from a given organism into a vector that can replicate in E. coli but not in the organism of interest; introduction into the cloned gene of a transposon which expresses a marker that can be selected for in the organism of interest; and return of the insertionally mutagenized gene to the chromosome of the organism of interest by homologous recombination. We chose Tn5 seq113 for performing shuttle mutagenesis because it (1) endoces the neo gene that confers kanamycin resistance to both E. coli and mycobacteria, (2) permits one to select for insertions into DNA sequences cloned into plasmid vectors using its neomycin hyperresistance phenotype,14 (3) transposes preferentially into DNA sequences of high guanine plus cytosine content, (4) facilitates sequencing of the gene into which it inserts owing to the presence of T7 and SP6 primer binding sites, and (5) lacks the cryptic gene encoding streptomycin resistance of Tn5, an important biohazard consideration for M. tuberculosis strains.
Selection for Transpositions into Cloned Genes: Neomycin Hyperresistance Selection 1. Transform any cloned mycobacterial gene into ec2270 (E. coli strain X2338::Tn5 seql) and select for colonies resistant to kanamycin and another antibiotic A (the genetic marker present on the plasmid cloning vector). 2. Inoculate individual colonies into LB (Luria broth) medium adjusted t2 G. V. Kalpana, B. R. Bloom, and W. R. Jacobs, Jr., Proc. Natl. Acad. Sci. U.S.A. 88,
5433 (1991). J3 D. K. Nag, H. V. Huang, and D. E. Berg, Gene 64, 135 (1988). 14 D. E. Berg, M. A. Schmandt, and J. B. Lowe, Genetics 105, 813 (1983).
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GENETIC SYSTEMS FOR MYCOBACTERIA
A
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MycobacterialBene i. o - - E . coil : : T n #
+
E. coti :: Tn 5
Neo Resistant
~¢~
Transforminto Select for Kan r
Plasmid :: Tn 5
Sinsie
duplication
Kanr colony
Homologous Recombination
'
I Double
mpla~ment
Kanr colony
FIG. 1. (A) Targetedshuttlemutagenesisof a mycobacterialgene usingTn5 seql. Randomshuttlemutagenesisof mycobacterialgenes.
(B)
to pH 7.2 and containing antibiotic A but no kanamycin and incubate overnight. 3. Plate dilutions of overnight cultures onto LB agar plates containing neomycin at 250/zg/ml plates and incubate for 24 hr. 4. Isolate plasmids from colonies that have grown on the neomycin plates. Retransform an E. coli cloning strain selecting for resistance to both antibiotic A and kanamycin to enrich for plasmids containing Tn5 seql.
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Ubraryof mycobacterialDNA in an E. coli plasmidvector I Transform into mycobacteda Select for Karlr
A
P b
l GeneRePlacement1 A
Mutation in Gene A
B
~F
C
Mutation in Gene B
Mutation in Gene C
FIG. 1. (continued).
5. Map the position of Tn5 seql insertion by standard restriction analyses.
Random Shuttle Mutagenesis of Mycobacteria The method described above can be used to isolate Tn5 seql insertions into any cloned DNA fragment. The description below provides a method to obtain random insertional mutants of M. smegmatis. This can be achieved by mutagenizing genomic library cloned mycobacterial DNA fragments in E. coli and then reintroducting these mutagenized DNA fragments into the chromosome of M. smegmatis. 1. In order to minimize the possibility of obtaining transposon insertions in the vector portion of recombinant mycobacterial clones a
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derivative of pBR322,15 pYUB36,1~ was constructed in which the nonessential 1.9-kb EcoRv to PoulI fragment has been deleted. Chromosomal DNA of M. smegmatis strain, mc26, was prepared as described above and partially digested with MspI. MspI DNA inserts of 4 to 7 kb were isolated by electroelution. The size-selected inserts were ligated to ClaI (a unique site)-digested pYUB36. The ligated DNAs were electroporated into ec2270 and plated on L agar containing both ampicillin and kanamycin at 40 and 50 /zg/ml, respectively. About 30,000 individual kanr and Ampr transformants were pooled and samples diluted 10-3 into 20 independent 5-ml L broth containing ampiciUin (no kanamycin) and incubated at 37° overnight. A 200 /zl sample from each culture yielded approximately 1000 neomycin hyperresistant colonies on L agar containing 250/.~g/ml neomycin, which selects for colonies resulting from transposition Tn5 seql into plasmids. Plasmid DNA from the combined pool of neomycin-resistant colonies was retransformed into ×2338. After 1 hr incubation, the transformants were directly inoculated into l-liter L broth containing kanamycin and ampicillin, grown to saturation at 37°, and plasmid DNA was prepared. This Tn5 seql-mutagenized plasmid library was then electroporated into M. smegmatis strain, mc26, and kanamycin-resistant transformants were selected on K agar [Middlebrook 7HI0 agar supplemented with 5 mg/ml casamino acids (Difco), 100/xg/ml diaminopimelic acid, 50/zg/ml thymidine, 40/~g/ml uracil, and 133 /zg/ml adenosine]. About 800 individual M. smegmatis transformants were screened for auxotrophy by streaking onto modified minimal Sauton medium TM without asparagine and this procedure yielded three auxotrophs. 12
Construction of Mycobacterial Genomic Libraries in Shuttle Cosmids Shuttle cosmids have been constructed that contain an origin of replication that functions in E. coli, an origin of replication that functions in mycobacteria, a kanamycin-resistance gene that functions in both E. coli and mycobacteria, bacteriophage h cos sequence that permits these mole15 F. Bolivar, et al., Gene 2, 95 (1977). 15a L. G. Wayne and G. A. Diaz, J. Bacteriol. 93, 1374 (1967).
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BamHI
II :l"l
BstE,
cos
neo
pYUB18 12 Kb
Sca '
P15A ~
orl
~ ~
Nhel
Xba
I
from the M. fortuitum plasmid pAL5000
FIG. 2. Escherichia c o l i / m y c o b a c t c r i a shuttle cosmid.
cules to be packaged into bacteriophage h heads, and unique restriction sites for construction of the genomic libraries. We chose to make mycobacterial genomic libraries in shuttle cosmid vector for the following reasons: the large insert size allows for the genome to be represented in as few as 100 clones, the large insert size provides the possibility of cloning sets of genes responsible for biosynthesis of complex polysaccharides or lipids, and the libraries can be stored stably as phage lysates following in vivo cosmid packaging. The vector pYUB 18 was constructed by inserting the BclI-BgllI fragment of bacteriophage h into pYUB125 (Fig. 2). This vector retains a unique BamHI site to be used for ligating Sau3A-digested chromosomal DNAs. In order to prevent the formation of unstable cosmids resulting from the ligation of multiple cosmid vectors within a larger recombinant cosmid, we have found it best to both gel purify large insert fragments for cloning and to treat the vector with alkaline phosphatase prior to ligation with the insert. Following in vitro packaging, the constructed libraries are transduced into cosmid in vivo packaging strains to permit amplification and efficient repackaging of recombinant cosmids into bacteriophage h heads thus allowing for storage of the libraries as phage lysates. 1. Digest CsCl-purified pYUB 18 DNA with BamH 1, phenol-chloro-
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form extract, and ethanol precipitate, and resuspend the DNA pellet in a volume of TE which gives a DNA concentration of 2 /zg//zl. 2. Treat the vector with alkaline phosphatase as follows: add 45 lzg BamHl-cut pYUB18 DNA to each of three tubes containing phosphatase buffer (Boehringer Mannheim) and add three different concentrations of calf intestinal alkaline phosphatase (CIAP) (Boehringer Mannheim) to final concentrations of 0.016, 0.008, and 0.004 units CIAP//~g pYUB18. Incubate for 2 hr at 37°. The efficiencies of the phosphatase reactions should be tested by setting up self-ligations at a concentration of 10 ng//zl of the three different CIAP reactions and of nonphosphatased BamHl-cut pYUBI8. Then 10 ng amounts of the ligations should be used to transform E. coli, and the number of transformants from each phosphatased ligation should be compared to the number of transformants from 10 ng of the unphosphatased BamHl-cut pYUB18. Use the phosphatased pYUB18 which gives approximately 1% self-ligation compared to the unphosphatased control. 3. Set up atrial Sau3A digests of approximately 300/xg of mycobacterial high molecular weight chromosomal DNA in five tubes, the first of which contains 100 /xg and the other four tubes which contain 50/~g each. Dilute Sau3A from New England Biolabs from its original concentration of 5 to 2.5 units//zl in 1X medium salt buffer. Add 1/zl of the diluted enzyme to the first tube of chromosome such that the final concentration of Sau3A is 0.025 units//~g DNA. Transfer 50% of the volume of tube 1 into tube 2 and mix such that the concentration of Sau3A in tube 2 is 0.0125 units//~g, and then serially transfer 50% of each tube until the last transfer is made into tube 5. Incubate the reactions at 37° for 30 min. The partial digestion reaction should be stopped by adding EDTA to a final concentration of 20 mM to each tube. 4. Load 2/~1 of each reaction tube onto a 0.4% agarose gel next to h DNA cut with either ApaI which gives two bands, 38.4 and 10 kb, orXhoI which gives two bands, 33.5 and 15 kb, to determine which partial digest gave the most 35-45 kb pieces of chromosomal DNA. Usually, reaction tubes 2 and 3 give the optimal DNA sizes. Pool the two tubes which give the most 35-45 kb DNA fragments, and load onto a 0.4% preparative agarose gel containing ethidium bromide. The gel should be made up in autoclaved 0.5X TBE buffer to reduce the possibility of nuclease contamination. Run the gel overnight at 20 V. Photograph and then cut out the gel slice that
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5.
6.
7.
8.
9.
10.
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contains DNA sized at 33.5 kb and larger, and place into a 2.5-cm wide dialysis bag containing 0.5-1.0 ml 0.5X autoclaved TBE buffer. Place the dialysis bag in the gel chamber such that the gel slice is pushed against the side of the bag closest to the cathode, and turn the voltage to 100 V to electroelute the DNA from the gel slice to the opposite side of the dialysis bag. Use a handheld UV lamp to monitor the progress of the ethidium-bromide-stained-DNA out of the gel slice. Once the DNA migrates to the opposite side of the dialysis bag, switch the positive and negative electrodes and run the voltage at 100 V for only a minute or two until the DNA just migrates away from the side of the dialysis tubing and enters the TBE solution in the bag. Pour the TBE and DNA solution out of the tubing and wash the tubing with 0.5X TBE to retrieve any solution that may have lingered. Gently phenol-chloroform extract and ethanol precipitate the DNA. Approximately 30/~g DNA, with an average size of 40 kb, should be recovered. Ligate the 40-kb chromosomal pieces with Sau3A ends to the BamHl-digested, phosphatased pYUB18. Ligations should have a 1 : 1 molar ratio of insert : vector, so add three times as much insert DNA as vector; 250 ng of B a m H 1-cut phosphatased pYUB 18 should be added to 750 ng Sau3A-cut 40-kb pieces of mycobacterial chromosome in a 20/.d ligation reaction whose final concentration is >50 ng//~l DNA. Add 400 units of T4 DNA ligase to the ligation reaction, incubate at 16° overnight, or at room temperature for 3 hr. In vitro package 4/~1 of the completed ligation reaction using Giga Pack Plus from Stratagene. Transduce the in vitro packaged mycobacterial cosmid library lysate into the in vivo packaging strains of E. coli, X2764, or X2819.16 To five separate tubes, add 0.1 ml of the lysate to 0.2 ml of saturated E. coli culture, incubate standing at 30° for 25 min, add 0.7 ml L broth, incubate shaking at 30° for 1 hr, and plate 0.2 ml per plate of L agar containing 25/~g/ml kanamycin. Incubate at 30 °. The in vitro packaged lysate should yield 5,000-10,000 transductants. Representative recombinant cosmids from a pYUB 18: M. tuberculosis DNA genomic library are shown in Fig. 3. To amplify the cosmid library, the transductants should be pooled,
16 W. R. Jacobs, J. F. Barrett, J. E. Clark-Curtiss, and R. Curtiss III, Infect. lmmun. 52, 101 (1986).
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2
3
4
5
6
7
8
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9
10
11
kb 23 9.9 6.6 4.4 2,3
2.0
¸
FIG. 3. Plasmid DNA isolated from E. coli containing random PYUB18:: M. tuberculosis DNA recombinant cosmids was digested with PstI and run on an agarose gel strained with ethidium bromide.
and inoculated into L broth containing 25 /zg/ml kanamycin to grow for in vivo packaging. In vivo packaging is performed as described. 16 Library lysates made in this manner yield titers of approximately 10~° transducing particles per milliliter and can be stored at 4° indefinitely. 11. To prepare the plasmid form of mycobacterial cosmid library DNA for electroporation of mycobacterial strains, the in oioo packaged lysate should be diluted in phage buffer and transduced into an E. coli cloning host to yield a minimum of 1000 kanamycin-resistant transductants. This library should be pooled, grown in liquid media containing kanamycin, 17 and plasmid DNA isolated by standard alkaline lysis methods. This library of plasmid DNA can be efficiently electroporated into M. smegmatis or other myobacteria using protocols described above.
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[25] G e n e t i c S y s t e m s for M y c o b a c t e r i a
By WILLIAM R. JACOBS, JR., GANJAMV. KALPANA, JEFFREY D. CIRILLO, LISA PASCOPELLA, SCOTT B. SNAPPER, RUPA A. UDANI, WILBUR JONES, RAI3L G. BARLETTA, a n d BARRY R. BLOOM
Introduction The ability to perform genetic analyses on bacteria has provided powerful tools and experimental systems to unravel fundamental biological processes. The advances of recombinant DNA technologies have ignited the development of genetic systems for bacteria that are difficult to work with. Clearly, the genus Mycobacterium contains a set of the most difficult bacterial species to manipulate experimentally. Despite its discovery over 100 years ago, the leprosy bacillus, Mycobacterium leprae, remains unable to be cultivated in the laboratory except in mouse footpads or in the ninebanded armadillo. Although Robert Koch pioneered the work of pure culture with Mycobacterium tuberculosis, the ability to extend genetic analyses beyond these initial studies has been greatly hampered by the slow growth of mycobacteria and the stubborn tendency to clump. The tuberculosis vaccine strain, BCG (bacille Calmette Gu6rin) has been used to vaccinate more individuals than any other live bacterial vaccine, yet little is known about mycobacterial gene structure and expression. The recent development of phage,l'2 plasmid,2 and gene replacement3 systems for the introduction of recombinant DNA into mycobacteria has opened up a new era of research on members of the genus Mycobacterium. Indeed, the use of these technologies should pave the way for the elucidation of mycobacterial virulence determinants and a detailed understanding of the mechanisms of drug action and resistance, as well as the development of recombinant multivalent BCG vaccines 1'4 capable of engendering immune responses to a variety of viral, bacterial, or parasitic antigens. The goal of this chapter is to bring together an array of recently developed methods and techniques which enable researchers to genetically manipulate mycobacterial species. I W. R. Jacobs, Jr., M. Tuckman, and B. R. Bloom, Nature (London) 327, 532 (1987). 2 S. B. Snapper, L. Lugosi, A. Jekkel, R. Melton, T. Kieser, B. R. Bloom, and W. R. Jacobs, Jr., Proc. Natl. Acad. Sci. U.S.A. 8S, 6987 (1988). 3 R. N. Husson, B. E. James, and R. A. Young, J. Bacteriol. 172, 519 (1990). 4 B. R. Bloom, J. Immunol. 10, i (1986).
METHODS IN ENZYMOLOGY, VOL. 204
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Mycobacterial Strains The mycobacteria fall into two general categories based on their growth rates. The fast-growing mycobacteria, consisting of such strains as M. smegmatis, M. phlei, M. aurum, and M. fortuitum, have doubling times of 2-3 hr and will yield a colony from a single cell in 3-4 days. In contrast, the slow-growing mycobacteria such as M. tuberculosis, BCG, or M. avium strains double every 18-26 hr and thus yield colonies from single cells in 14-28 days. Although it is likely that the vectors and methodologies described here are applicable to many mycobacterial species, we have focused our efforts on the fast-growing M. smegmatis and the slow-growing BCG. The strains used are (I) M. smegmatis strain mcZ6, a single colony isolate of the predominant colony type found in the ATCC 607 culture; (2) M. smegmatis strain mc2155, an efficient transformation mutant 5 of mc26; and (3) BCG-Pasteur obtained as a lyophilized pellet from the Statens Serum Institute in Copenhagen, Denmark.
Biohazard Considerations Biosafety level 3 (BL3) containment facilities and corresponding practices and equipment are appropriate for activities involving the propagation and manipulation of M. tuberculosis and M. boris; all other mycobacterial species including BCG and M. avium can be safely handled in BL2 containment facilities. 6 Experiments involving the introduction of recombinant mycobacterial DNA into Escherichia coli or any mycobacterial host require that the investigator obtain approval from the Institution's Biosafety Committee before the initiation of experiments. 7 The National Institutes of Health Recombinant DNA Advisory Committee (RAC) recommends that experiments involving the introduction of recombinant DNA from a class 3 agent, such as M. tuberculosis, into a class 2 agent such as M. smegmatis be performed using BL3 containment facilities. The deliberate transfer of a gene conferring drug resistance whose acquisition could compromise the use of a drug to control the pathogen, such as the introduction of isoniazid resistance into M. tuberculosis, can only be
5 S. B. Snapper, R. E. Melton, S. Mustafa, T. Kieser, and W. R. Jacobs, Jr., Mol. Microbiol. 4, 1123 (1990). 6 U.S. Dept. of Health and Human Services, CDC/NIH. "Biosafety in Microbiological and Biomedical Laboratories." U.S. Government Printing Office, Washington, D.C. HHS Publication No. (CDC) 86-8395 (1986). 7 U.S. Department of Health and Human Services, Federal Register 51, 16957 (1986).
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performed following approval from both the RAC and the Institutional Biosafety Committee.
Growth and Maintenance of Mycobacterial Strains
Media For Growth of Mycobacteria in Liquid Culture M - A D C - T W broth: Dissolve 4.7 g of Difco (Detroit, MI) Middlebrook 7H9 broth base and 2 ml glycerol in 900 ml deionized water. Autoclave 20 min. After cooling, add 100 ml albumin-dextrose complex (ADC) enrichment and 2.5 ml of 20% Tween 80 solution. The albumin component of the ADC enrichment is essential for the growth of BCG but is not needed for M. smegmatis growth. ADC enrichment: Dissolve 2 g glucose, 5 g bovine serum albumin fraction V, (Boehringer Mannheim, Indianapolis, IN), and 0.85 g NaC1 in 100 ml deionized water. Filter sterilize and store at 4 °. 20% Tween 80: Add 20 ml of polyoxyethylene sorbitan monooleate to 80 ml deionized water, heat at 55 ° for 30 min to dissolve completely, and filter sterilize.
For Colony Titrations, Transformations, and Transductions with Shuttle Phasmids Middlebrook 7H10 agar: Add 19 g Middlebrook 7H10 agar to 900 ml deionized water, autoclave 20 rain, and allow to temper to 55 °. Add 100 ml ADC enrichment and pour 40-45 ml per plate. Cycloheximide can be added at a final concentration of 50 t~g/ml to inhibit mold contamination. Kanamycin is added to a final concentration of 10 t~g/ml for the selection of shuttle phasmid transductants or transformants. For Propagating Mycobacteriophage and Shuttle Phasmids. It is most important not to have Tween 80 in media used to propagate phage. DB+ bottom agar: Add 20 g Dubos oleic acid agar base, 1 g NaCI, and 7.5 g glucose to 1 liter deionized water. Autoclave 20 rain, temper to 55 °, and add MgSO4 and CaCI2 to final concentrations of 10 and 2 raM, respectively. Mycobacteriophage top agar: Add 0.5 g Middlebrook 7H9 broth base, 0.1 g NaCI, 0.75 g glucose, and 0.7 g agar to 100 ml deionized water. For BCG, supplement this by adding 1.0 g proteose peptone #3
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(Difco), 0.5 g yeast extract (Difco), and 5 ml of a 20% glycerol solution (v/v).
Growth of Mycobacterium smegmatis and BCG The slow growth of the mycobacteria necessitates that excellent aseptic technique be followed since most contaminants will outgrow mycobacteria, particularly the slow-growing mycobacteria. Since the time required for a single cell to yield a colony might be up to 3 to 6 weeks, which is often essential for genetic analyses, agar plates onto which bacteria cells are plated must be poured thick and wrapped with Parafilm both to prevent desiccation of the media and to reduce possible risks of mold contamination. Tween 80 is an essential component of mycobacterial media that reduces the clumping of mycobacteria and aids in the preparation of single cell suspensions. However, microscopic analyses will usually demonstrate clumps of mycobacterial cells even when the cultures are grown in Tween 80. Mycobacterium smegmatis will clump less than most of the other fastgrowing mycobacteria and can yield a reasonably satisfactory single cell suspension when grown in M-ADC-TW broth, in baffled flasks, with shaking at 37°. In contrast, BCG will clump considerably if grown like M. smegmatis. We have found it best either to grow BCG standing, for phage work, or to grow it in tissue culture roller bottle flasks to yield high levels of reasonably unclumped viable cells. Mycobacteriophage and Shuttle Phasmids Historically, mycobacteriophage have been used to type various mycobacterial isolates. Novel phage vectors, termed shuttle phasmids, have been constructed from the mycobacteriophage TM4 and L1.1'2 Shuttle phasmids s can replicate in mycobacteria as phage capable of undergoing lysis or lysogeny. These vectors can also replicate in E. coli as cosmids and, thus, be packaged into bacteriophage h heads to facilitate subsequent cloning of additional genes. 8
Preparation of High-Titer Lysates of Mycobacteriophage and Shuttle Phasmids 1. Prepare M. smegmatis mc26 cells for a phage infection by inoculating 0.1 ml of the starter culture into 25 ml of M-ADC-TW broth in a 250-ml baffled flask. Shake the flask at 100 rpm on a platform 8 W. R. Jacobs, Jr., S. B. Snapper, M. Tuckman, and B. R. Bloom, Rev. Infect. Dis. 11, $404 (1989).
[25]
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shaker at 37° overnight. Although mycobacteriophage infections are inhibited by Tween 80, overnight cultures of M. smegmatis contain Tween hydrolase that destroys most of the added Tween. However, the addition of Tween 80 is still necessary to yield an unclumped lawn of mycobacterial cells. We have found it best for BCG phage lawns to use 10-day-old standing cultures of BCG grown at 37° from a 1 : 1000 dilution of an exponential BCG culture. 2. Dilute the phage or shuttle phasmid, 10-fold serially, in MP buffer (10 mM Tris, pH 7.6, 100 mM NaC1, 10 mM MgSO 4 , 2 mM CaCI 2) to obtain approximately 5 × 104 to 105 plaque forming units (pfu) per milliliter. 3. Mix 2.0 ml of mc26 cells ( - 3 x 108 cells/ml) with 1.0 ml of the phage or shuttle phasmid diluted to 5 x 104 pfu/ml in a sterile 13 x 100 mm culture tube. Incubate at 37° for 30 min to allow adsorption to Occur.
4. Pipette 0.3 ml of the phage-cell mixture to a culture tube containing 3.0 ml of DB~b top agar from M. smegmatis or DB~b supplemented top agar for BCG, mix the phage-cell suspension with the top agar by gently rotating the tube in the palms of your hands, and aseptically pour on top of a DBth plate. Allow the agar to solidify. Invert the plate and incubate at 37° for 24-36 hr for M. smegmatis or 7-10 days for BCG. For each lysate, we normally prepare five plates. 5. Pipette 5 ml of MP buffer on each plate. Set plates upright at 4 ° overnight. 6. Pipette off as much liquid as possible (usually - 3 ml) from each plate lysate and combine all of the lysate fluids in a 50-ml polypropylene tube. Centrifuge the lysates at 4° at 3000 g for 10 rain. Filter the supernatant fluid through a 0.45-/zm filter unit. Transfer the filtered phage lysate preparation to a sterile phage bottle. Store at 4 °. Note: Do not add chloroform to mycobacteriophage lysates as most mycobacteriophage contain lipids and are inactivated by nonpolar solvents.
Phage Purification and Isolation of DNA Mycobacteriophage can be easily purified and concentrated on CsCl density gradients as described for E. coli phage.9 The procedures described here have given high yields of phage particles from mycobacteriophage D29, L1, TM4, and shuttle phasmid derivatives. DNA can be efficiently 9 R. W. Davis, D. Botstein, and J. R. Roth, "Advanced Bacterial Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980.
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isolated from the purified phage particles following proteinase K treatment, phenol-chloroform extractions, and ethanol precipitations.
Transfection of Phage DNA by Electroporation 1. Transfect phage DNA by electroporation. A detailed protocol for electroporation of mycobacteria is described below. Basically, the same procedure is carried out for transfection with phage DNA. The following considerations should be taken into account for transfection protocols: to maximize the yield of plaques, it is essential to dilute the cell suspension after electroporation, and incubate further for I hr at 37°. The dilution is important to reduce the level of harmful chemicals (radicals) generated during electroporation, and the subsequent incubation allows the mycobacterial cells to recover from the electric shock. 2. Mix 0.1 ml of an appropriate dilution of the electroporation mixture with 0.2 ml of a freshly grown culture (logarithmic to early stationary culture) of mycobacterial cells. Then add 3 ml of soft agar and plate. Usually, transfection frequencies around 105 pfu//zg of phage DNA are obtained.
Construction of Shuttle Phasmids Shuttle phasmids are chimeric constructs between a mycobacteriophage and an E. coli plasmid, in which the plasmid is inserted in a nonessential region of the phage genome. Usually, an E. coli cosmid is used as a plasmid, since the possibility of performing in vitro packaging greatly facilitates the construction of recombinant structures. The steps involved in making such constructs are as follows: 1. Ligate mycobacteriophage DNA at high DNA concentrations (-> 100 /zg/ml, usually between 250 and 500/xg/ml). 2. Digest the ligated mycobacteriophage DNA partially with a frequent cutter (e.g., Sau3AI), so that the majority of the fragments obtained are in the range of 44 kilobases (kb) (if the cosmid used is - 6 kb, like pHC79). At this stage it is possible to fractionate the 44-kb fragments if desired. 3. Linearize the cosmid vector DNA with a compatible restriction enzyme (usually the corresponding hexamer, e.g., BamHI); treat with alkaline phosphatase if desired. 4. Ligate the linearized cosmid vector with the digested mycobacteriophage DNA from Step 2 at high DNA concentrations (250-500 /zg/ml) so that concatemeric DNA is obtained, with intermingled fragments of mycobacteriophage and cosmid DNA. 5. In vitro package, using commercially available packaging mixes.
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7.
8.
9.
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The ligated DNA is packaged in h particles by virtue of the cos sites present in the cosmid vector. At this step a transducing phage lysate containing the recombinant DNA is obtained. With the phage lysate transduce an appropriate E. coli host containing mutations in the hsdR, mcrA, and mcrB genes, such as ER1451 (New England Biolabs, Beverly, MA). Select transductants containing recombinant cosmid DNA by plating on appropriate antibiotic plates (L agar plus 25/zg/ml of ampicillin for pHC79). Pool transductants and isolate plasmid DNA by standard procedures. The plasmids isolated from this pool represent an insertion library of the cosmid vector in the mycobacteriophage genome. Only those plasmids with insertions within a dispensable region of the mycobacteriophage genome will give rise to shuttle phasmids. Electroporate an appropriate mycobacterial host (mc26 or mc2155 for L1 and TM4), using the pool of recombinant plasmids, as previously described. Screen the mycobacteriophage plaques obtained using the cosmid vector as probe. Positive hybridizing plaques represent true shuttle phasmids. At this stage, we have observed with the construction of TM4-derived shuttle phasmids that almost all the resulting plaques have lost the insertion of the cosmid DNA (1 positive for every 400 plaques), presumably the result of a recombination event between multiple recombinant cosmid::phage molecules entering a cell and recombining. Shuttle phasmids can be isolated, ligated, in vitro packaged into bacteriophage h heads, and transduced into E. coli cells where they replicate as cosmids conferring antibiotic resistance.
Cloning Genes in Shuttle Phasmids
Unique cloning sites within the cosmid vector can be useful for cloning foreign DNA, provided that they are absent from the phage genome. For example, both TM4 and L1 lack EcoRI and HindlII sites within the phage genome, and therefore the unique EcoRI site or HindlII sites in pHC79 can be used to introduce additional foreign DNA. The size of foreign DNA that can be cloned in a shuttle phasmid is constrained by the size requirements of the mycobacteriophage packaging machinery, as well as by the cosmid packaging mechanism. In this way, additional genes, 2 to 4 kb in size, can be efficiently cloned into shuttle phasmids. The procedure can be summarized as follows: 1. Ligate shuttle phasmid DNA so that long concatemers are obtained. 2. Digest shuttle phasmid DNA with an appropriate restriction enzyme (e.g., EcoRI for L1 shuttle phasmids) to yield large DNA vector
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molecules that can be ligated to a gene of choice with compatible restriction enzyme ends. . After ligation, in vitro package, and proceed as described above for the construction of shuttle phasmids. By using the temperate shuttle phasmid phAE15, 2 derived from mycobacteriophage LI, one can select or screen for mycobacterial colonies carrying the recombinant DNA with the gene of interest.
E l e c t r o p o r a t i o n o f Mycobacterium smegmatis a n d B C G
The highest efficiencies for transformation of mycobacteria have been obtained using electroporation. We have found that mycobacteria can withstand very high voltages for extended periods of time. Thus, a highresistance electroporation buffer such as 10% glycerol is used along with a high parallel resistance. This method results in long time constants, which have been shown to give the highest efficiencies of transformation. The protocol which we use is similar to that used for E. coli.10 1. Inoculate 1 liter of M - A D C - T W broth with 10 ml of a 10-to 15-day culture of BCG (A600 = 0.5-1.0). In the case ofM. smegmatis, 1 liter of M - A D C - T W broth can be directly inoculated from a frozen stock or colony directly. 2. Incubate at 37 ° until the A600reaches 0.5-1.0. For BCG this period varies greatly, from 7 to 25 days, depending on the passage of the culture and growth conditions; M. smegmatis, however, is almost always at the correct stage of growth by 48 hr. 3. Harvest in 250-ml centrifuge bottles. Centrifuge for I0 min at 10,000 rpm and 4 °. Discard supernatant, 4. Resuspend each BCG pellet in 5 ml of ice-cold 10% glycerol, pool them into two 15-ml polypropylene conical tubes, and centrifuge for 10 min at 3000 rpm and 4 °. Mycobacterium smegmatis, however, should be directly suspended in the centrifuge bottles in 250 ml of 10% glycerol and centrifuged as before. 5. Resuspend the BCG pellets in I0 ml cold 10% glycerol. At this point, M. smegmatis pellets may be resuspended in 10 ml cold 10% glycerol, pooled into two 50-ml polypropylene conical tubes, and the volume raised to 50 ml with 10% glycerol. Centrifuge for I0 min at 3000 rpm and 4°. 6. Repeat the wash in Step 5. 7. Resuspend each pellet to a final volume of 1 ml in 10% glycerol. l0 W. J. Dower, J. F. Miller, and C. W. Ragsdale, Nucleic Acids Res. 16, 6127 (1988).
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1 I. 12.
13.
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The protocol can be continued with the freshly prepared cells as shown below, or, at this step, the cells can be aliquoted into Eppendorf tubes and frozen quickly in dry ice-ethanol to be used later by thawing slowly at room temperature (sometimes the thawed cells will contain salts and cause arcing; this can be prevented by washing the cells in the Eppendorf tubes with 10% glycerol once or twice). Place DNA (5 pg-5/zg; 5 gl volume, maximum) and 50 tzl of cells in an Eppendorf tube and mix well by pipetting up and down. Place on ice 1 min. Set the voltage of the pulser (Bio-Rad, Richmond, CA) to 2500 V, 25/zF, and the pulse controller (Bio-Rad) to 1000 ohms. Sometimes the cells will arc with a high parallel resistance; washing the cells further with 10% glycerol will usually prevent this. If DNA containing high salt is added, either a lower resistance in the pulse controller must be used or the DNA must be ethanol precipitated and washed with 70% (v/v) ethanol at least once. If a lower resistance is used, lower transformation frequencies will result; therefore, ethanol precipitation is recommended. Transfer the solution containing the cells and DNA into a cuvette with a 0.2-cm electrode gap (Bio-Rad). Tap the cuvette against the bench several times to get the cells to the bottom of the cuvette and to remove as many bubbles as possible. Place the cuvette in the pulser and expose to one pulse (time constants are usually between 15.0 and 25.0 msec). For BCG electroporations add 0.4 ml of M - A D C - T W medium directly to the cuvette and suspend the cells well in it. For M. smegmatis add 1 ml of M - A D C - T W broth, suspend the cells in it, and transfer to a round-bottomed 15-ml polypropylene tube. Incubate at 37° approximately 3 hr to allow antibiotic expression. Plate the cells on selective medium.
Both the M. smegmatis and BCG electroporation procedures are extremely sensitive to the concentration of salts present in the DNA sample used. The DNA used should be washed with 70% ethanol if there is any possibility of the presence of excess salt. Also, it is important to add as small a volume of DNA as possible to prevent dilution of the bacterial cell concentration in the cuvette. Ligations must be ethanol precipitated and washed with 70% ethanol before they can be used (it is possible to use small amounts of a ligation, but significantly lower transformation efficiencies will result). After the last wash, if the packed cell volume is not very close to or is more than 1 ml, do not raise the volume to 1 ml final,
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just add enough 10% glycerol to allow the cells to be pipetted easily. Fresh cells in our hands give transformation efficiencies above 106 transformants/ /zg (using a 10-kb plasmid), and frozen cells give efficiencies which vary from 104 to 106 transformants//zg. Chromosomal DNA Isolation and Plasmid Isolation from Mycobacterial Cells
Isolation of High Molecular Weight Mycobacterial Chromosomal DNA Using Mini-Beadbeater Cell Disruptor 1. Grow 100 ml of culture ofM. smegmatis or BCG cells to approximately 3 x 108 cells/ml. Normally, we use overnight M. smegmatis cultures started from a 0.1-ml starter culture or 7- to 9-day-old BCG cultures that have been grown with shaking. Large numbers of freshly grown ceils are critical to obtaining good yields of high molecular weight DNA. 2. Pellet the cells by centrifuging in 50-ml polypropylene tubes in a Sorvall RT6000 centrifuge at 3000 rpm for 10 min at 4°. 3. Carefully, pour off the supernatant fluid and resuspend the pellet in 0.5 ml of homogenization buffer (0.3 M Tris, pH 8, 0.1 M NaCI-6 m M EDTA). 4. Transfer the resuspended cells to conical 2-ml screw-cap vial which is one-fourth to one-half filled with 0.5-mm acid-washed sterile glass beads. 5. Place the vial on the Mini-Beadbeater cell disruptor (Biospec Products, BartlesviUe, OK) and beat it for 1 rain. 6. Transfer the entire contents of the tube to a 15-ml conical polypropylene tube. Wash the 2-ml Beadbeater vial with 1 ml of homogenization buffer and add this to the disrupted cell suspension. 7. Extract with an equal volume of phenol-chloroform (1 : 1) solution. Use Tris-buffered neutralized phenol. Repeat the extraction one more time. After each extraction, spin the tubes in a Sorvall RT6000 (Dupont, Wilmington, DE) centrifuge at 3000 rpm for 10 min. 8. Peform one chloroform-isoamyl (24-1) extraction and then ethanol precipitated with 1/10 volume of 3 M sodium acetate, pH 5.0, and 2 volumes of cold ethanol. 9. Place the tubes at - 7 0 ° for 30 min or - 2 0 ° overnight for ethanol precipitation. 10. Spin the tubes for 10-15 min at 3000 rpm in Sorvall RT6000 centrifuge.
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GENETIC SYSTEMS FOR MYCOBACTERIA
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11. Pour off the ethanol and wash the pellet with 2 ml of cold 70% ethanol. 12. Spin the precipitate again for 5 min in a SorvaU RT6000 centrifuge at 3000 rpm. 13. Pour off the ethanol and dry the pellet. 14. Resuspend the pellet in I ml of TE (10 mM Tris-1 mM EDTA) buffer. Do not shake or vortex vigorously. Let the tube sit for a few minutes and then just gently mix up and down 2-3 times. If the pellet still does not dissolve, add more TE. 15. Digest RNA by adding RNase A at I00 /zg/ml using 10 mg/ml boiled stock. Incubate at 37° for 30 min. 16. Extract with an equal volume of phenol-chloroform twice as before. 17. Extract with an equal volume of chloroform-isoamyl alcohol once. 18. Ethanol precipitate with 1/I0 volume of 3 M sodium acetate and 2 volumes of ethanol. 19. Pellet the DNA, pour offthe supernatant fluid, dry, and resuspend the pellet in 1 ml TE.
Plasmid DNA Isolation from Mycobacterial Cells We have adapted the alkaline lysis protocol 1~with the following modifications: 1. Grow 5 to 10 ml of the mycobacterial cell culture to approximately saturation (overnight for the fast growers, several days for slow growers). 2. Harvest the cells by centrifugation, resuspend the pellet in 150/xl of GTE (25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM glucose) containing 10 mg/ml of lysozyme, and incubate at 37° overnight. 3. Add 2 volumes (300/zl) of fresh 0.2 M N a O H - I % sodium dodecyl sulfate (SDS) and mix by inversion. Incubate at 4 ° for 10 min. 4. Add 1.5 volumes (225 /zl) of 5 M potassium acetate and mix by inversion. Incubate at 4 ° for 10 min. 5. Centrifuge in microcentrifuge for 30 min to 1 hr. This step is critical to obtain a good plasmid preparation. 6. Remove and extract the supernatant with an equal volume of CPI (chloroform-phenol-isoamyl alcohol, 24 : 25 : 1). 7. Transfer the aqueous phase to a clean tube and add 2 volumes (1200/zl) of room temperature ethanol, incubate for 5 min at room temperature, spin down the DNA pellet, wash with 70% ethanol, and dry briefly under reduced pressure. u H. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979).
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8. Resuspend in 25 to 50/zl of TE buffer, then incubate at 65° for 15 min. Insertional Mutagenesis A method of generating random chromosomal mutations by inserting a piece of DNA containing a selectable marker gene is essential for the mycobacteria since it permits one to select mutants arising from a single mutagenic event directly, even from a pool of clumped cells. Insertional mutagenesis using transposons has been a valuable method for mutational analysis. In the absence of a mycobacterial transposon that transposes at high frequency we have developed an indirect method of random mutagenesis, a random shuttle mutagenesis. 12 This method was developed by extending the strategy of targeted mutagenesis (Fig. 1). Shuttle mutagenesis of a targeted gene involves three steps: cloning of the desired gene from a given organism into a vector that can replicate in E. coli but not in the organism of interest; introduction into the cloned gene of a transposon which expresses a marker that can be selected for in the organism of interest; and return of the insertionally mutagenized gene to the chromosome of the organism of interest by homologous recombination. We chose Tn5 seq113 for performing shuttle mutagenesis because it (1) endoces the neo gene that confers kanamycin resistance to both E. coli and mycobacteria, (2) permits one to select for insertions into DNA sequences cloned into plasmid vectors using its neomycin hyperresistance phenotype,14 (3) transposes preferentially into DNA sequences of high guanine plus cytosine content, (4) facilitates sequencing of the gene into which it inserts owing to the presence of T7 and SP6 primer binding sites, and (5) lacks the cryptic gene encoding streptomycin resistance of Tn5, an important biohazard consideration for M. tuberculosis strains.
Selection for Transpositions into Cloned Genes: Neomycin Hyperresistance Selection 1. Transform any cloned mycobacterial gene into ec2270 (E. coli strain X2338::Tn5 seql) and select for colonies resistant to kanamycin and another antibiotic A (the genetic marker present on the plasmid cloning vector). 2. Inoculate individual colonies into LB (Luria broth) medium adjusted t2 G. V. Kalpana, B. R. Bloom, and W. R. Jacobs, Jr., Proc. Natl. Acad. Sci. U.S.A. 88,
5433 (1991). J3 D. K. Nag, H. V. Huang, and D. E. Berg, Gene 64, 135 (1988). 14 D. E. Berg, M. A. Schmandt, and J. B. Lowe, Genetics 105, 813 (1983).
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GENETIC SYSTEMS FOR MYCOBACTERIA
A
549
MycobacterialBene i. o - - E . coil : : T n #
+
E. coti :: Tn 5
Neo Resistant
~¢~
Transforminto Select for Kan r
Plasmid :: Tn 5
Sinsie
duplication
Kanr colony
Homologous Recombination
'
I Double
mpla~ment
Kanr colony
FIG. 1. (A) Targetedshuttlemutagenesisof a mycobacterialgene usingTn5 seql. Randomshuttlemutagenesisof mycobacterialgenes.
(B)
to pH 7.2 and containing antibiotic A but no kanamycin and incubate overnight. 3. Plate dilutions of overnight cultures onto LB agar plates containing neomycin at 250/zg/ml plates and incubate for 24 hr. 4. Isolate plasmids from colonies that have grown on the neomycin plates. Retransform an E. coli cloning strain selecting for resistance to both antibiotic A and kanamycin to enrich for plasmids containing Tn5 seql.
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OTHER BACTERIAL SYSTEMS
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Ubraryof mycobacterialDNA in an E. coli plasmidvector I Transform into mycobacteda Select for Karlr
A
P b
l GeneRePlacement1 A
Mutation in Gene A
B
~F
C
Mutation in Gene B
Mutation in Gene C
FIG. 1. (continued).
5. Map the position of Tn5 seql insertion by standard restriction analyses.
Random Shuttle Mutagenesis of Mycobacteria The method described above can be used to isolate Tn5 seql insertions into any cloned DNA fragment. The description below provides a method to obtain random insertional mutants of M. smegmatis. This can be achieved by mutagenizing genomic library cloned mycobacterial DNA fragments in E. coli and then reintroducting these mutagenized DNA fragments into the chromosome of M. smegmatis. 1. In order to minimize the possibility of obtaining transposon insertions in the vector portion of recombinant mycobacterial clones a
[25]
GENETIC SYSTEMS FOR MYCOBACTERIA
2.
3. 4. 5.
6.
7.
8.
9.
10.
551
derivative of pBR322,15 pYUB36,1~ was constructed in which the nonessential 1.9-kb EcoRv to PoulI fragment has been deleted. Chromosomal DNA of M. smegmatis strain, mc26, was prepared as described above and partially digested with MspI. MspI DNA inserts of 4 to 7 kb were isolated by electroelution. The size-selected inserts were ligated to ClaI (a unique site)-digested pYUB36. The ligated DNAs were electroporated into ec2270 and plated on L agar containing both ampicillin and kanamycin at 40 and 50 /zg/ml, respectively. About 30,000 individual kanr and Ampr transformants were pooled and samples diluted 10-3 into 20 independent 5-ml L broth containing ampiciUin (no kanamycin) and incubated at 37° overnight. A 200 /zl sample from each culture yielded approximately 1000 neomycin hyperresistant colonies on L agar containing 250/.~g/ml neomycin, which selects for colonies resulting from transposition Tn5 seql into plasmids. Plasmid DNA from the combined pool of neomycin-resistant colonies was retransformed into ×2338. After 1 hr incubation, the transformants were directly inoculated into l-liter L broth containing kanamycin and ampicillin, grown to saturation at 37°, and plasmid DNA was prepared. This Tn5 seql-mutagenized plasmid library was then electroporated into M. smegmatis strain, mc26, and kanamycin-resistant transformants were selected on K agar [Middlebrook 7HI0 agar supplemented with 5 mg/ml casamino acids (Difco), 100/xg/ml diaminopimelic acid, 50/zg/ml thymidine, 40/~g/ml uracil, and 133 /zg/ml adenosine]. About 800 individual M. smegmatis transformants were screened for auxotrophy by streaking onto modified minimal Sauton medium TM without asparagine and this procedure yielded three auxotrophs. 12
Construction of Mycobacterial Genomic Libraries in Shuttle Cosmids Shuttle cosmids have been constructed that contain an origin of replication that functions in E. coli, an origin of replication that functions in mycobacteria, a kanamycin-resistance gene that functions in both E. coli and mycobacteria, bacteriophage h cos sequence that permits these mole15 F. Bolivar, et al., Gene 2, 95 (1977). 15a L. G. Wayne and G. A. Diaz, J. Bacteriol. 93, 1374 (1967).
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BamHI
II :l"l
BstE,
cos
neo
pYUB18 12 Kb
Sca '
P15A ~
orl
~ ~
Nhel
Xba
I
from the M. fortuitum plasmid pAL5000
FIG. 2. Escherichia c o l i / m y c o b a c t c r i a shuttle cosmid.
cules to be packaged into bacteriophage h heads, and unique restriction sites for construction of the genomic libraries. We chose to make mycobacterial genomic libraries in shuttle cosmid vector for the following reasons: the large insert size allows for the genome to be represented in as few as 100 clones, the large insert size provides the possibility of cloning sets of genes responsible for biosynthesis of complex polysaccharides or lipids, and the libraries can be stored stably as phage lysates following in vivo cosmid packaging. The vector pYUB 18 was constructed by inserting the BclI-BgllI fragment of bacteriophage h into pYUB125 (Fig. 2). This vector retains a unique BamHI site to be used for ligating Sau3A-digested chromosomal DNAs. In order to prevent the formation of unstable cosmids resulting from the ligation of multiple cosmid vectors within a larger recombinant cosmid, we have found it best to both gel purify large insert fragments for cloning and to treat the vector with alkaline phosphatase prior to ligation with the insert. Following in vitro packaging, the constructed libraries are transduced into cosmid in vivo packaging strains to permit amplification and efficient repackaging of recombinant cosmids into bacteriophage h heads thus allowing for storage of the libraries as phage lysates. 1. Digest CsCl-purified pYUB 18 DNA with BamH 1, phenol-chloro-
125]
GENETIC SYSTEMS FOR MYCOBACTERIA
553
form extract, and ethanol precipitate, and resuspend the DNA pellet in a volume of TE which gives a DNA concentration of 2 /zg//zl. 2. Treat the vector with alkaline phosphatase as follows: add 45 lzg BamHl-cut pYUB18 DNA to each of three tubes containing phosphatase buffer (Boehringer Mannheim) and add three different concentrations of calf intestinal alkaline phosphatase (CIAP) (Boehringer Mannheim) to final concentrations of 0.016, 0.008, and 0.004 units CIAP//~g pYUB18. Incubate for 2 hr at 37°. The efficiencies of the phosphatase reactions should be tested by setting up self-ligations at a concentration of 10 ng//zl of the three different CIAP reactions and of nonphosphatased BamHl-cut pYUBI8. Then 10 ng amounts of the ligations should be used to transform E. coli, and the number of transformants from each phosphatased ligation should be compared to the number of transformants from 10 ng of the unphosphatased BamHl-cut pYUB18. Use the phosphatased pYUB18 which gives approximately 1% self-ligation compared to the unphosphatased control. 3. Set up atrial Sau3A digests of approximately 300/xg of mycobacterial high molecular weight chromosomal DNA in five tubes, the first of which contains 100 /xg and the other four tubes which contain 50/~g each. Dilute Sau3A from New England Biolabs from its original concentration of 5 to 2.5 units//zl in 1X medium salt buffer. Add 1/zl of the diluted enzyme to the first tube of chromosome such that the final concentration of Sau3A is 0.025 units//~g DNA. Transfer 50% of the volume of tube 1 into tube 2 and mix such that the concentration of Sau3A in tube 2 is 0.0125 units//~g, and then serially transfer 50% of each tube until the last transfer is made into tube 5. Incubate the reactions at 37° for 30 min. The partial digestion reaction should be stopped by adding EDTA to a final concentration of 20 mM to each tube. 4. Load 2/~1 of each reaction tube onto a 0.4% agarose gel next to h DNA cut with either ApaI which gives two bands, 38.4 and 10 kb, orXhoI which gives two bands, 33.5 and 15 kb, to determine which partial digest gave the most 35-45 kb pieces of chromosomal DNA. Usually, reaction tubes 2 and 3 give the optimal DNA sizes. Pool the two tubes which give the most 35-45 kb DNA fragments, and load onto a 0.4% preparative agarose gel containing ethidium bromide. The gel should be made up in autoclaved 0.5X TBE buffer to reduce the possibility of nuclease contamination. Run the gel overnight at 20 V. Photograph and then cut out the gel slice that
554
OTHER BACTERIAL SYSTEMS
5.
6.
7.
8.
9.
10.
[25]
contains DNA sized at 33.5 kb and larger, and place into a 2.5-cm wide dialysis bag containing 0.5-1.0 ml 0.5X autoclaved TBE buffer. Place the dialysis bag in the gel chamber such that the gel slice is pushed against the side of the bag closest to the cathode, and turn the voltage to 100 V to electroelute the DNA from the gel slice to the opposite side of the dialysis bag. Use a handheld UV lamp to monitor the progress of the ethidium-bromide-stained-DNA out of the gel slice. Once the DNA migrates to the opposite side of the dialysis bag, switch the positive and negative electrodes and run the voltage at 100 V for only a minute or two until the DNA just migrates away from the side of the dialysis tubing and enters the TBE solution in the bag. Pour the TBE and DNA solution out of the tubing and wash the tubing with 0.5X TBE to retrieve any solution that may have lingered. Gently phenol-chloroform extract and ethanol precipitate the DNA. Approximately 30/~g DNA, with an average size of 40 kb, should be recovered. Ligate the 40-kb chromosomal pieces with Sau3A ends to the BamHl-digested, phosphatased pYUB18. Ligations should have a 1 : 1 molar ratio of insert : vector, so add three times as much insert DNA as vector; 250 ng of B a m H 1-cut phosphatased pYUB 18 should be added to 750 ng Sau3A-cut 40-kb pieces of mycobacterial chromosome in a 20/.d ligation reaction whose final concentration is >50 ng//~l DNA. Add 400 units of T4 DNA ligase to the ligation reaction, incubate at 16° overnight, or at room temperature for 3 hr. In vitro package 4/~1 of the completed ligation reaction using Giga Pack Plus from Stratagene. Transduce the in vitro packaged mycobacterial cosmid library lysate into the in vivo packaging strains of E. coli, X2764, or X2819.16 To five separate tubes, add 0.1 ml of the lysate to 0.2 ml of saturated E. coli culture, incubate standing at 30° for 25 min, add 0.7 ml L broth, incubate shaking at 30° for 1 hr, and plate 0.2 ml per plate of L agar containing 25/~g/ml kanamycin. Incubate at 30 °. The in vitro packaged lysate should yield 5,000-10,000 transductants. Representative recombinant cosmids from a pYUB 18: M. tuberculosis DNA genomic library are shown in Fig. 3. To amplify the cosmid library, the transductants should be pooled,
16 W. R. Jacobs, J. F. Barrett, J. E. Clark-Curtiss, and R. Curtiss III, Infect. lmmun. 52, 101 (1986).
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GENETIC SYSTEMS FOR MYCOBACTERIA
1
2
3
4
5
6
7
8
555
9
10
11
kb 23 9.9 6.6 4.4 2,3
2.0
¸
FIG. 3. Plasmid DNA isolated from E. coli containing random PYUB18:: M. tuberculosis DNA recombinant cosmids was digested with PstI and run on an agarose gel strained with ethidium bromide.
and inoculated into L broth containing 25 /zg/ml kanamycin to grow for in vivo packaging. In vivo packaging is performed as described. 16 Library lysates made in this manner yield titers of approximately 10~° transducing particles per milliliter and can be stored at 4° indefinitely. 11. To prepare the plasmid form of mycobacterial cosmid library DNA for electroporation of mycobacterial strains, the in oioo packaged lysate should be diluted in phage buffer and transduced into an E. coli cloning host to yield a minimum of 1000 kanamycin-resistant transductants. This library should be pooled, grown in liquid media containing kanamycin, 17 and plasmid DNA isolated by standard alkaline lysis methods. This library of plasmid DNA can be efficiently electroporated into M. smegmatis or other myobacteria using protocols described above.
