Vol. 27, No. 2INFECTION AND IMMUNITY, Feb. 1980, p. 368-3750019-9567/80/02-0368/08$02.00/0

Isolation of Deoxyribonucleic Acid from Mycobacteriumavium by Rapid Nitrogen Decompression

PAUL M. YANDELL AND CHARLOTTE McCARTHY*Biology Department, New Mexico State University, Las Cruces, New Mexico 88003

Deoxyribonucleic acid (DNA) of high molecular weight could be isolated fromcells of Mycobacterium avium if the cells were exposed to nitrogen gas at 1,500lb/in2 for 30 min and then brought to atmospheric pressure by rapid decompres-sion. DNA isolated from the cells had a molecular weight of 4.8 x 106 to 17.4 x106. DNA was also released into the fluid in which the cells were suspendedduring nitrogen decompression. One-half of this DNA, representing 3% of thetotal DNA phosphorus in the cells had a uniform molecular weight of 4.2 x 106.This DNA was linear in conformation, and removal of associated carbohydratesdid not change its sedimentation rate. The biological function or significance ofthe 4-megadalton DNA was not determined.

Mycobacterium avium produces several dif-ferent colony types on 7H10 agar (24), whichmay be cultured and maintained in a relativelystable condition. Schaefer et al. (22) demon-strated that a nonpigmented colony form,termed transparent, was pathogenic for animals,whereas the opaque variant derived from it wasnot. The cellular constituents of the two colonyforms that cause the difference in pathogenicityhave not been determined. As yet no geneticsystem has been found for M. avium; however,several years ago, McCarthy (17) reported thatthe rate oftransition from the transparent (path-ogenic) to the opaque (nonpathogenic) colonytype was too great to be explained on the basisof mutation. This plus the apparent irreversibil-ity of the transition invoked the explanation thatextragenetic information, possibly a plasmid,was responsible for the transparent colony formand its concurrent pathogenicity.A major obstacle in testing this hypothesis

has been the intractability of the cells with re-gard to gentle lysis, which is the first step re-quired for the isolation and analysis of the de-oxyribonucleic acid (DNA) of an organism. Pro-cedures for the release of DNA from mycobac-teria, especially pathogens with slow rates ofgrowth, are very inefficient. Most protocols re-quire, after the cells have been harvested, longtime periods for the lytic or breakage process,and this increases the likelihood that undesira-ble changes in the DNA will occur.Many investigators have used delipidation

and then grinding with glass beads to mechani-cally break the cells. This type of extraction wasemployed by Chargaff and Saidel (2), who ac-complished the isolation of nucleoproteins fromavian tubercle bacilli in 1949. A total of 1 week

was expended to release the nucleic acid and topartially concentrate it before the actual chem-ical analyses were begun.The combined use oflysozyme and detergents,

as exemplified by the Marmur procedure (16),has proven to be effective for the lysis of manybacteria and the gentle release of DNAs havinghigh molecular weights. However, this methodis ineffective for the isolation of DNA frommycobacteria because of their resistance to theaction of lysozyme and detergents. Mizuguchiand Tokunaga (21) demonstrated that Mycobac-terium smegmatis, after incubation in 0.1 Mglycine for 2 h, was made susceptible to lysis bylysozyme. Simpson and Wayne (23) modifiedthis procedure to lyse mycobacteria having slowgrowth rates by increasing the incubation timeand the concentration of glycine. The logarith-mic-phase cells were incubated with 0.2 M gly-cine overnight, and a second overnight incuba-tion with lysozyme completed the lysis of thecells. This method was reported to be successfulfor obtaining DNA from refractory mycobacte-ria, such as M. avium. However, these authorsdid not comment on the colony form of M.avium that responded to this treatment.During the past 10 years one of us (C.M.) has

attempted to extract DNA from the transparentcolony type of M. avium by several methods.These have included delipidation, grinding withglass beads, a variety of detergents or solubi-lizing agents, and preincubation in glycine, su-crose, and lysozyme. Each of these procedureshas proven to be unsatisfactory since it wasrarely, if ever, possible to obtain an extract fromsuch treated cells which contained enough ma-terial to form strings ofDNA upon precipitationin cold ethanol.

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DNA FROM M. AVIUM BY NITROGEN DECOMPRESSION 369

We have recently experimented with the useof nitrogen decompression ofM. avium and havefound that this weakens the cells sufficiently sothat lysis and DNA extraction can be achievedby rapid, conventional means. Partial character-ization of a DNA species with a uniform molec-ular weight that was released from M. avium bynitrogen decompression is also included in thisreport.

MATERIALS AND METHODSBacterial strains. M. avium strain DM 9 is a

transparent colony variant (serotype 1) and has beenstudied previously (18-20). A thymine-requiring strainof Escherichia coli JC411 with the ColEl plasmid wasobtained from Stanley Falkow.Chemicals and isotopes. [nP]monopotassium

phosphate (100 mCi/mmol), [2-'4C]thymine (53 mCi/mmol), and aquasol scintillation cocktail were pur-chased from New England Nuclear Corp. Agarosebeads (Bio-Gel A-15m; exclusion limit, 1.5 x 107 dal-tons; 100 to 200 mesh) were purchased from Bio-RadLaboratories.Media and culture conditions. Concentrated fro-

zen stocks of M. avium, which were maintained at-40'C, were thawed, and 0.1 ml (which containedabout 109 colony-forming units per ml) was inoculatedinto 1 liter ofBOACGG medium in a 2.8-liter low-formculture flask. The BOACGG medium contained B salts(20), oleic acid-albumin complex (20), 0.5% glucose,0.5% glycerol, and 0.1% Tween 80 (BBL MicrobiologySystems). The flasks were incubated at 370C for 10 to14 days to a concentration of 5 x 10" colony-formingunits per ml.

For radioactive labeling of M. avium DNA, smallcells (length, 1 Mim) were obtained by selective filtra-tion as described by McCarthy (18), and these wereinoculated into tris(hydroxymethyl)aminomethane(Tris)-buffered BOACGG medium with 0.1 mMKH2PO4 and 0.4 tuCi of [3P]monopotassium phosphateper ml. The Tris-buffered BOACGG medium was pre-pared from B salts that lacked the phosphate salts,and it was supplemented with 10 mM Tris-hydrochlo-ride buffer (pH 7). The cultures, which were initially105 colony-forming units per ml, were incubated at370C and 150 rpm on a rotary shaker to a cell densityof 3 x 108 colony-forming units per ml.

Cells were harvested by centrifugation and washedwith 10 mM Tris-hydrochloride (pH 7) and 0.1%Tween 80, and the pellets were stored at -20°C in 50-ml plastic centrifuge tubes (Falcon Plastics).

Viability determinations for M. avium were madeon Middlebrook and Cohn 7H10 agar (BBL Microbi-ology Systems).

E. coli JC411 (ColEl) was cultured and labeled with['4C]thymine as described by Clewell and Helinski (3).

Nitrogen gas decompression. An M. avium cellpellet in a 50-ml conical plastic centrifuge tube wasthawed and suspended in 5 to 15 ml of 50 mM Tris-hydrochloride-50 mM ethylenediaminetetracetate,pH 8; sucrose at 25% was included unless otherwiseindicated. The tube was inserted into a hollowed-outarea of a small piece of styrofoam, and this was placedin a cell disruption bomb (model 4635; Parr Instrument

Co.), which was partially filled with ice. The dischargetube was inserted into the cell suspension, and the topof the bomb was clamped on. Nitrogen gas was intro-duced into the bomb over a period of 2 to 5 min, andthe pressure was maintained at 1500 lb/in2 for 30 minunless otherwise stated.The release of the cells was sometimes explosive

and was therefore carried out in a safety hood. Al-though subsequent treatment usually entailed centrif-ugation, a centrifuge tube was of the conformationthat the entire sample would impact on the bottom ofthe tube and explode upward and out in all directions.Therefore, the discharge tube from the bomb wasplaced about one-third of the depth into a 250-mlErlenmeyer flask which was placed on ice within abeaker. Disposable gloves were worn, and aluminumfoil was used to envelope the discharge tube and tocover the flask during decompression. Upon de-compression, which occurred within 30 to 60 s, thecells became distributed over the bottom of the flaskwith little loss of sample. The cells were treated asindicated below for the individual experiments andthen transferred to a centrifuge tube. The glassware,disposable gloves, and aluminum foil were decontam-inated by autoclaving. The discharge tube was decon-taminated by flushing it with 200 ml of 10% formal-dehyde solution, and it was then thoroughly rinsedwith distilled water.

Preparation of TCL. Lysozyme at 170 ,ug/ml wasadded to bomb-treated cells of M. avium, and themixture was maintained on ice for 30 min. Triton X-100 was added to a final concentration of 5%, and themixture was placed on ice with occasional swirling foran additional 30 min. The resulting preparation wascentrifuged at 38,000 x g for 25 min at 4°C. Thesupernatant fraction, designated the Triton-clearedlysate (TCL), was decanted from the pellet and eithermaintained on ice or stored at -20°C.

Cleared lysates were prepared from E. coli as de-scribed by Katz et al. (14).

For some procedures, DNA in a TCL was concen-trated by polyethylene glycol precipitation, as de-scribed by Humphreys et al. (12).DNA extraction from bomb-treated cells. The

TCL pellets were suspended in 150 mM sodium chlo-ride-15 mM sodium citrate, and sodium dodecyl sul-fate was added to a final concentration of 0.4%. After30 min on ice, the mixture was brought to roomtemperature and extracted by the Marmur procedure(16), including ribonuclease treatment and a final pre-cipitation in isopropanol.Column chromatography. DNA released into the

TCL ofM. avium was separated from ribonucleic acid,protein, and detergent by passage through a column(2.5 by 28 cm) of Bio-Gel A-15m agarose beads. Thecolumn was standardized before use with blue dextranand human hemoglobin.Chemical procedures. DNA phosphorus (DNA-

P) was determined by the method of Burton (1), usingdeoxyadenosine monophosphate as a standard; be-cause only purine-bound deoxyribose reacts with thisassay, values were multiplied by two to determinetotal DNA-P. Total carbohydrate was determined asdescribed by Dubois et al. (7), using glucose as astandard. Protein was estimated by the method of

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370 YANDELL AND McCARTHY

Lowry et al. (15), using bovine albumin as a standard;the assay was modified by heating the cells in 0.5 NNaOH at 90'C for 10 min before the addition of theother reagents. DNA from M. avium was separatedfrom polysaccharides by precipitation with cetyltri-methyl ammonium bromide (CTAB) precipitation, asdescribed by Hill et al. (11).

Sucrose density gradients. Linear sucrose gra-dients of 5 to 20% (wt/vol) were constructed by usinga Beckman density gradient former. The linearity ofthe gradients was confirmed by refractometer mea-surements of tube fractions. Neutral gradients wereprepared in 100 mM NaCl-50 mM phosphate, pH 6.7;alkaline gradients were in 1 M NaCl-1 mM ethylene-diamine tetraacetate-300 mM NaOH.A sample of 0.2 ml that contained 1 to 5 ,tg ofDNA

was layered onto a 4.8-ml sucrose gradient, and thiswas centrifuged in an SW65 rotor at 150,000 x g in aBeckman model L or L2B centrifuge at 10'C for thetimes indicated below. Fractions of about 0.2 ml werecollected from the bottom of the tube into scintillationvials which, after addition of aquasol, were counted ina Packard Tri-Carb liquid scintillation spectrometer.The 18S open circle form of ColE1 was used as astandard, and molecular weights were calculated fromthe formulas described by Clowes (4) for linear, opencircular, and covalently closed duplexes.Dye-buoyant density equilibrium centrifuga-

tion. Solutions that contained 0.1 ml of ethidiumbromide (6 mg/nl), 1.61 ml of saturated CsCl (1.85 g/ml) in 20mM Tris-hydrochloride (pH 8.5), and enoughDNA to bring the solution to 3 ml were mixed in acentrifuge tube. The mixture was overlaid with 2 ml oflight mineral oil and centrifuged in an SW65 rotorat 44,000 rpm for 60 h at 150C in a Beckman modelL2B ultracentrifuge. Fractions were collected, and 20-pd samples were removed to scintillation vials. Aquasolwas added, and the samples were counted in a PackardTri-Carb liquid scintillation spectrometer. Peak frac-tions were pooled, extracted twice with an equal vol-ume of CsCl-saturated isopropanol to remove theethidium bromide, and dialyzed at 4°C before analysison sucrose gradients.

RESULTS

Cell damage caused by nitrogen de-compression. Cells of M. avium that had beenstored at -20°C were thawed and suspended inTris buffer. Samples of 5 ml were distributedinto each of four 50-ml plastic centrifuge tubes,and these were individually exposed to 1,500 lb/in2 of nitrogen gas pressure for 0, 15, 30, or 45min. The treated cells were released within 30 sthrough the discharge tube and centrifuged at18,000 x g for 30 min at 40C to remove themodified cells. Cellular damage was assessed bythe amount of material in the supernatant frac-tions that absorbed at 260 nm. The exposure ofM. avium to the high gas pressure, followed byrapid decompression, resulted in release of 260-nm-absorbing material, and a nearly maximumvalue was achieved after a 15-min exposure (Fig.

I5 30 45MINUTES

FIG. 1. Effect of decompression on M. avium cellsexposed to nitrogen gas for different time intervals. Acell pellet of DM9 was suspended in 50 mM Tris-hydrochloride-50 mM ethylenediaminetetraacetate,pH 8, and distributed in 5-ml samples (1.4 x 1010colony-forming units per ml) to plastic, conical cen-trifuge tubes. Individual cell suspensions wereplacedin a cell disruption bomb and exposed to 1,500 lb/in2of nitrogen gas pressure for different time intervals.The nitrogen-treated cells were released within 30 sthrough the discharge tube to centrifuge tubes stand-ing on ice. The bomb-treated cells were centrifugedat 18,000 x g for 30 min at 4°C to clear the superna-tant fluid of whole cells and cellular debris. Thesupernatant fractions were then assayed for 260-nm-absorbing material.

1). The bombed cells were microscopically indis-tinguishable from untreated cells, and about 58%remained viable after exposure to nitrogen gasfor 30 min.

Different batches of cells decompressed after30 min of exposure to nitrogen gas at 1500 lb/in2and the respective supernatant fluids were as-sayed for DNA content. In general, there was atotal of 30 to 40 ttmol of DNA-P in the cellsconcentrated from 1 liter of medium, and theproportion of cell protein (in micrograms permilliliter) to DNA (in micromoles of phosphorusper milliliter) was 1,000:1. Approximately 4% ofthe total cell DNA-P was released into the su-pernatant fluid by rapid decompression. If thebombed cells were additionally treated with ly-sozyme at 170 jig/ml and Triton X-100 at 5%,about 7% of the total cell DNA-P was released.Only 0.5% of the cell DNA-P was released bylysozyme and Triton X-100 treatment of cellsthat had not undergone nitrogen decompression.DNA purification. In the studies described

below, the cells were suspended in buffer thatcontained 25% sucrose. These were treated withnitrogen gas at 1,500 lb/in2 for 30 min, rapidlydecompressed, and then treated with lysozymeand Triton X-100. The preparation was centri-fuged at 38,000 x g for 25 min. The resultingsupernatant fluid was the TCL, and the pellet

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DNA FROM M. AVIUM BY NITROGEN DECOMPRESSION 371

that contained the modified cells was referred toas the TCL pellet.As stated above, we were unable to obtain

significant amounts ofDNA from M. avium cellsunless they were treated by nitrogen decompres-sion, and therefore we have nothing with whichto compare our procedure. Extraction of DNAfrom the TCL pellet was achieved by the pro-cedure outlined by Marmur (16). That is, themodified cells were susceptible to lysozyme, so-dium dodecyl sulfate, sodium perchlorate, andchloroform-isoamyl alcohol treatment, and theaqueous extracts that were ultimately producedcontained material that precipitated in cold 95%ethanol. This material was treated with ribonu-clease, deproteinized, and finally purified bywinding the precipitate onto a glass rod fromisopropanol as described by Marmur (16). Useof this method provided purified high-molecu-lar-weight DNA that represented 8 to 12% of theoriginal DNA-P of the unmodified cells. ThisDNA was designated the chromosomal DNA.We performed a preliminary examination of

the DNA that was released into the TCL. Be-cause of the explosiveness with which the cellswere released from the bomb, it was initiallyassumed that the material represented onlysmall fragments. Another possibility was that itcontained plasmid DNA, since Triton X-100 hadbeen used in conjunction with other techniquesto purify plasmid DNA from chromosomal DNAin E. coli (14). To distinguish between these twopossibilities, a TCL of M. avium which con-tained 6.9% of the total DNA in the cell suspen-sion was chromatographed on agarose beadshaving an exclusion limit of 1.5 x 107 daltons.The fractions were assayed for absorbency at260 nm, and two peaks were resolved (Fig. 2).The first peak (fractions 11 to 15) correspondedto material that eluted slightly after the voidvolume. This peak contained 3.3% of the totalDNA-P in the original unmodified cell suspen-sion. It was designated TCL DNA and was sep-arated by column chromatography from othercellular constituents, as well as from the sub-stances added during extraction, all of whicheluted together in the large second peak.Determination of the molecular weight.

To demonstrate that the chromosomal DNAwas of high molecular weight and to ascertainmore carefully the molecular weight of the TCLDNA, 33P was used to label both of these sub-stances. The TCL [nP]DNA and the chromo-somal [33P]DNA were prepared and subjectedto zonal centrifugation on neutral sucrose gra-dients. ColEl [14C]DNA was included as a stan-dard. The profiles obtained are shown in Fig. 3.Zonal centrifugation of the ColEl [14C]DNA

FRACTION NUMBER

FIG. 2. Chromatography of a TCL ofM. avium onan agarose gel column. An 8-ml sample ofa TCL waseluted from a Bio-Gel A-15m column with 50 mMpotassium phosphate, pH 6.8. The absorbance of the2.5-ml fractions at 260 nm is indicated. Elution ofthestandards is indicated by the arrows. bd, Blue dex-tran (2 x 106 daltons); hh, human hemoglobin (6.6 x104 daltons).

yielded two peaks that corresponded to the 18Sopen circular form and the 24S covalently closedcircular DNA-protein complex (Fig. 3A). Thechromosomal DNA (Fig. 3B) of M. avium washeterogeneous in size, with sedimentation coef-ficients varying from 16.1S to 27.3S. These coef-ficients corresponded to molecular weights of 4.8and 17.4 megadaltons, respectively. The TCLDNA from M. avium was uniform in size andhad a sedimentation coefficient of 15.1S (Fig.3B). The molecular weight of this DNA wascalculated to be 4.2 megadaltons, if linear inconformation.Dye-buoyant density equilibrium centrif-

ugation. To determine the conformation of theTCL [33P]DNA, it was concentrated by poly-ethylene glycol precipitation and centrifuged toequilibrium in CsCl density gradients which con-tained ethidium bromide. Fractions were thencollected to determine the location of the 33P-labeled material. The results are shown in Fig.4 and indicate that two peaks were resolved.These were designated peak I and peak II, al-though the material in peak I (at the bottom ofthe tube) contained only one-tenth the 33P inpeak II. Because of the possibility that either ofthese might contain DNA in the closed circularconfiguration expected for plasmid DNA, thefractions from each of these peaks were pooledand subjected to centrifugation on alkaline su-crose gradients (Fig. 5). The 3P-labeled materialin peak II (Fig. 5B) sedimented on the alkalinesucrose gradient at the same rate as that ex-

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372 YANDELL AND McCARTHY IFC.IMN

22f

U 22

18

14

10

10 20 30 40 50

FRACTION NUMBER

FIG. 3. Sucrose gradient sedimentation at neutral

pH ofM. avium and ColEJ DNA. ['4C]DNA obtained

from an E. coli (CoIEJ) TCL by polyethylene glycol

precipitation was centrifuged on one gradient. M.

avium chromosomal [33P]DNA and TCL [33P]DNA

were centrifuged on separate gradients. The gra-

dients of 5 to 20%, sucrose were centrifuged at 150,000

x g in an SW65 rotor at 100C for 105 min. Fractions

were collected from the bottom of the tubes and as-

sayed for radioactivity. The direction of centrifuga-

tion was from right to left. (A) ColE) 18S DNA

(fractions 30 to 40). (B) Symbols: A, M. avium chro-

mosomal DNA; 40, M. avium TCL DNA.

pected for the TCL DNA, if in a linear confor-

mation. The material in peak I, however, was

hydrolyzed under alkaline conditions and re-

mained at the top of the gradient (Fig. 5A).

Thus, although two peaks were observed in the

dye-buoyant density gradients, only one (peak

II) contained DNA, and this material behaved

as though in a linear conformation.

DNA-associated carbohydrates. Hill et al.

(11) found that mycobacterial DNA isolated by

phenol extraction and ethanol precipitation was

often associated with large quantities of polysac-

charides. Therefore, these two DNA prepara-

tions from M. avium were tested for carbohy-

2= 32

1 ~~~~~~~~~~28U 6 24

5 20

4 16

3-12

2 8

1 4

5 15 20 25 30 35FRACTION NUMBER

FIG. 4. Ethidium bromide-cesium chloride densitygradient analysis of material from an M. avium TCL.33p-labeled material was concentrated from a TCLby polyethylene glycol precipitation. The concen-trated material was centrifuged at 44,000 rpm for 60h at 150C in an ethidium bromide-cesium chloridedensity gradient that contained 100 jig of ethidiumbromide per ml and 1 g of CsCl per ml. Fractionswere collected from the bottom of the tube, and 20 1dsamples of each were assayed for radioactivity. Thedirection of centrifugation was from right to left.Peak I occurred in fractions 2 to 5, and peak IIoccurred in fractions 11 to 13.

drates (7). It was found that the TCL DNAcontained four times and the chromosomal DNAcontained seven times more carbohydrate thancould be accounted for by the deoxyribose in theDNA. According to Hill et al. (11), absorbencyratios for solutions of mycobacterial DNA at 255nm/280 nm and at 255 nm/230 nm are lowerthan the expected values of 2.0 and 2.2, respec-tively. This was attributed to the high carbohy-drate content of mycobacterial DNA.Removal of polysaccharides from the chro-

mosomal DNA was followed directly by com-paring total carbohydrate content with the deox-yribose contributed by DNA and indirectly bynoting increases in absorbency ratios at 255 nm/280 nm and 255 nm/230 nm. Treatment with

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DNA FROM M. AVIUM BY NITROGEN DECOMPRESSION 373

(4

"0

2 l10 20 30 40 50FRACTION NUMBER

FIG. 5. Sucrose gradient sedimentation at alka-linepH ofpeaks obtained from an ethidium bromide-cesium chloride density gradient. Peak fractions fromthe ethidium bromide-cesium chloride density gra-dient illustrated in Fig. 4 were pooled separately,extracted with CsCl-saturated isopropanol to removeethidium bromide, and dialyzed at 40C against 50mM Tris-hydrochloride-50 mM ethylenediaminetet-raacetate,pH 8, to remove CsCl. These were analyzedon 5 to 20% sucrose gradients at alkaline pH alongwith [33P]DNA from an M. avium TCL. The gra-dients were centrifuged at 150,000 x g in an SW65rotor at 100C for 3 h. Fractions were collected fromthe bottom of the tubes and assayed for radioactivity.The arrows indicate the position to which the TCLDNA sedimented. The direction ofcentrifugation wasfrom right to left. (A) Peak I, fractions 2 to 5 of Fig.4. (B) Peak II, fractions 11 to 13 of Fig. 4.

CTAB by the procedure of Hill et al. (11) re-

sulted in the removal of 99% of the total carbo-hydrate in the DNA preparation, with a concom-itant recovery of nearly 60% of the total DNA(Table 1). This purification was mirrored byincreases in absorbance ratios.TCL DNA was also treated with CTAB to

determine whether such treatment wouldchange its apparent molecular weight. TCLDNA was split into three samples. One sample

TABLE 1. CTAB purification ofM. aviumchromosomal DNAAbsorbancy ratios Amt of

Amt of total

DNA sample' 255 255 DNA-P carbo-nm/280 nm/230 (Pmol) hydratenm rim

per mnl (Ag) perml

Before CTAB 1.72 1.81 0.148 185.6After CTAB 1.87 2.06 0.084 2.1

a DNA was isolated from a TCL pellet by the Mar-mur procedure as described in the text. Samples wereremoved from the DNA preparation before and afterCTAB purification for determination of absorbencyratios, amount of DNA-P per milliliter, and amount oftotal carbohydrate per milliliter.

was treated with CTAB to remove polysaccha-ride; this procedure included chloroform-iso-amyl alcohol and ethanol treatments. Therefore,DNA in the second sample was treated withchloroform-isoamyl alcohol and ethanol to de-termine whether these procedures, withoutCTAB precipitation, caused any change in sizeof the DNA. The third sample was untreated.The absorbance ratios at 255 nm/230 nm for theCTAB-treated sample and the nontreated con-trol were 1.82 and 1.24, respectively. The threesamples were centrifuged on sucrose gradients,and it was found that removal of the polysac-charides had no effect on the sedimentation ofthe DNA. Thus, association of the TCL DNAwith polysaccharides did not affect its molecularweight determination.

DISCUSSIONExplosive decompression has rarely been em-

ployed for breakage of cells, and the parametershave varied with the system under study. Asnoted by Coakley et al. (5), few studies havebeen made to determine the duration of theperiod cells should be under pressure. We havefound that nearly maximal damage occurs aftera 30-min exposure of M. avium to 1,500 lb/in2(Fig. 1), that release of some DNA occurs underthese conditions, and that the cells are suscep-tible to further lysis by detergents. The pressureof 1,500 lb/in2 used in this study corresponds to10.4 MN/M2 or 102 atmospheres and was chosenbecause this is at the intermediate level of thepressures of 3.5 to 35 MN/M2 reported in otherinvestigations (6).Rapid decompression as a method for break-

ing bacterial cells was described by Fraser (9),who found that nitrogen gas at 900 lb/in' causeda culture of E. coli to become 72% nonviableafter an exposure time of only 3 min. Hunterand Commerford (13) used an apparatus thatwas the prototype for the Parr disruption bomb,

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374 YANDELL AND McCARTHY

and they found that 900-lb/in' exposure for 20min homogenized rat liver tissue sufficiently sothat intact nuclei were obtained, whereas pres-sures above 1,300 lb/in2 caused disruption of thenuclei. Foster et al. (8) designed a chamberwhich contained a frangible disk. Cells weremaintained at 1,740 lb/in2 in the chamber for 75min, and then pressure was increased until thedisk burst and the expelled material was col-lected, using precautions adequate for dealingwith pathogens. Rupture efficiencies varied withthis apparatus, depending upon the microorga-nism tested, from only 10% rupture for Staphy-lococcus aureus to 59% for Serratia marcescens.

Fraser (9) theorized that cell breakage wasdue to expansion of the gas within the bacterialcell upon decompression. Hemmingsen andHemmingsen (10), however, dispute this becausein their investigation E. coli remained 100%viable after exposure to 300 atmospheres for 0.5h and subsequent rapid decompression. Theysuggested that mechanical forces generated asthe cell suspension was forced through an orificeat the time of decompression were responsiblefor the breakage process described by otherworkers.Although the mechanism of action is un-

known, the pretreatment of M. avium cells withnitrogen decompression permits the rapid isola-tion of high-molecular-weight DNA by the Mar-mur procedure. We extended this study in aninitial attempt to discover plasmids in this or-ganism; in the meantime, plasmids have beenisolated from strains of this species by anothertechnique (6). However, during this investiga-tion, a DNA species with a uniform size of 4megadaltons was obtained. The origin of the 4-megadalton DNA obtained in the TCL is notknown. It may represent pieces of chromosomalDNA that resulted from either mechanical shearor enzymic action. However, its facile releasefrom the cells is particularly difficult to explain.It was present in fluid released from the bombedcells and increased in amount upon treatment ofthe cells with detergent, and it was present whenthe preparation was maintained at 40C through-out the various procedures. An alternative ex-planation, therefore, would be that the 4-mega-dalton DNA was present in the cells before anyof the treatments. This is difficult to verify be-cause of our inability to specifically label theDNA of this organism. However, we are contin-uing an investigation of this material.We have repeatedly used nitrogen decompres-

sion for isolation of DNA from four differentstrains of M. avium and their various colonytypes and have found it to be a reliable, repro-ducible procedure. We believe that it may be

used effectively for the gentle, rapid isolation ofDNA from other members of the genus.

ACKNOWLEDGMENTS

This work was supported by Public Health Service researchgrants AI-11171 and AI-15293 from the National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED

1. Burton, K. 1956. A study of the conditions and mecha-nisms of the diphenylamine reaction for the colorimetricestimation of deoxyribonucleic acid. Biochem. J. 62:315-322.

2. Chargaff, E., and H. F. Saidel. 1949. On the nucleopro-teins of avian tubercle bacilli. J. Biol. Chem. 177:417-428.

3. Clewell, D. B., and D. R. Helinski. 1969. Supercoiledcircular DNA-protein complex in Escherichia coli: pu-rification and induced conversion to an open circularDNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166.

4. Clowes, R. C. 1972. Molecular structure of bacterialplasmids. Bacteriol. Rev. 36:361-405.

5. Coakley, W. T., A. J. Bater, and D. Lloyd. 1977.Disruption of micro-organisms. Adv. Microbiol. Physiol.16:279-341.

6. Crawford, J. T., and J. H. Bates. 1979. Isolation ofplasmids from mycobacteria. Infect. Immun. 24:979-981.

7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers,and F. Smith. 1956. Colorimetric method for determi-nation of sugars and related substances. Anal. Chem.28:350-356.

8. Foster, J. W., R. M. Cowan, and T. A. Maag. 1962.Rupture of bacteria by explosive decompression. J.Bacteriol. 83:330-334.

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