Vol. 163, No. 2

Chromosomal-DNA Amplification in Bacillus subtilisC. RON WILSON* AND ALICE E. MORGAN

Biotechnology Laboratory, The Dow Chemical Co., Midland, Michigan 48674

Received 15 February 1985/Accepted 1 May 1985

Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from BaciUus subtilisBC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetra-cycline. The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistanceafter 30 generations of growth in the absence of selection. Of the four isolates, three contained amplifiedchromosomal DNA closely associated with the Tetr phenotype. The intensity of restriction fragments present inHindIll and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence oftandemly duplicated DNA. Disparity in the size and number of amplified fragments suggested that thetandemly duplicated DNA is different in all three isolates. The sizes of the duplicated DNA present in RAD1,RAD6, and RAD7 were estimated to be 10, 19, and 20 kiobases, respectively. No amplified DNA was detectedin RAD2. Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) lociof RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus.Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus. Cotransfer of amplifiedDNA with the guaA locus or other nearby loci in the absence of tet was not observed. In every case, loss of Tetrwas accompanied by loss of amplified DNA. A possible explanation for the occurrence of the amplified DNA ispresented.

The occurrence of amplified sequences of chromosomalDNA in both eucaryotes and procaryotes is well docu-mented (1, 3-5, 11, 13, 14, 18). Chromosomal duplicationsoccur at high frequency. Anderson and Roth (5) estimatedthat 3% of a Salmonella typhimurium population carriedtandem duplications. The duplications occur spontaneouslyfrom illegitimate unequal recombination between nonhomo-logous sequences on homologous chromosomes or, morefrequently, legitimate unequal recombination between ho-mologous sequences located at different points on homolo-gous chromosomes (4, 5). Almost all duplications examinedhave been tandem arrays of a repeated unit (4). The repeatunit associated with an amplified region of DNA has aconstant size and defined endpoints for each isolate, but thesize and endpoints vary among isolates (3, 13). Tisty et al.(18) reported that 100 unstable lac+ revertants of a lacmutant contained amplified chromosomal DNA ranging insize from 10 to 30 kilobases. The amplified DNA associatedwith each isolate contained multiple copies (40 to 200) of thelac operon, but the repeated unit of DNA was different ineach case.

In many cases, chromosomal duplications are detected byincreased production of a selectable gene product (1, 14).Increased production of enzymes encoded by the lactose andhistidine operons resulting from gene amplification has beenreported (3, 7). Selection for increased resistance to anantibiotic has also been used to isolate strains containingamplified sequences of chromosomal DNA (20, 24). Selec-tion is based on the integration of plasmid DNA carryingantibiotic resistance into the bacterial chromosome. Integra-tion is accomplished by constructing a replication-defectiveplasmid which carries a segment of DNA homologous to thechromosome of the recipient organism. The site for plasmidintegration can be directed by the nature of the homologousDNA fragment inserted into the plasmid. Recently, therehave been several reports of such vehicles designed tointegrate into the chromosomes of Streptococcus pneumo-

* Corresponding author.

niae and Bacillus subtilis (12, 20, 24). After integration, thedegree of amplification of the resistance determinant iscontrolled by the level of antibiotic in the growth medium.

In this paper, we report on tetracycline (Tc)-resistant(Tetr) isolates of B. subtilis, all but one of which contain anamplified region of chromosomal DNA. These isolates differfrom those reported by Young (24) and Saito et al. (15) inthat they occur spontaneously after regeneration ofprotoplasts and their isolation does not require the integra-tion of plasmid DNA sequences. Transduction has been usedto map the Tc resistance (tet) locus close to the guaA locusand to the origin of replication of the B. subtilis chromo-some. In each case, the amplified DNA is closely linked toTetr. Loss of Tetr is accompanied by loss of amplified DNA.Differences in the size and number of amplified bandspresent in restriction digests indicate that the amplifiedchromosomal DNA is different in each isolate. The natureand stability of Tetr and amplified DNA present in theseisolates are discussed.

(Part of this research was presented at the AmericanSociety for Microbiology Conference on Genetics and Mo-lecular Biology of Industrial Organisms, Bloomington, Ind.,October 1984.)

MATERIALS AND METHODS

Bacterial strains. The strains of B. subtilis used in thisstudy are described in Table 1.Media and chemicals. The growth medium used for stabil-

ity studies and for DNA isolation was TSBS (Trypticase soybroth [BBL Microbiology Systems, Cockeysville, Md.] sup-plemented with 50 jig each of adenine, uracil, and thymineper ml). The medium used in transduction experiments wasthat described by Anagnostopoulos and Spizizen (2) modi-fied based on the marker being selected and the nutritionalrequirements of the recipient. Unless otherwise specified, allchemicals and antibiotics were purchased from Sigma Chem-ical Co., St. Louis, Mo.

Restriction endonucleases. Restriction enzymes were pur-chased from either Bethesda Research Laboratories,

445

JOURNAL OF BACTERIOLOGY, Aug., 1985, p. 445-4530021-9193/85/080445-09$02.00/0Copyright © 1985, American Society for Microbiology

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446 WILSON AND MORGAN

TABLE 1. Strains of B. subtilisAnti-

Strain Genotype biotic Source or referenceresis-tance

BC92 puraA16 argA ile none Steve PhillipsIA177 guaAl rplV Eryr Bacillus Genetic Stock

CenterQB928 aroI906 purB33 dal-I none Bacillus Genetic Stock

trpC2 CenterQB944 purA16 cysAJ4 trpC2 none Stan ZahlerRADM purAl6 argA ile tet Tetr This workRAD2 purAJ6 argA ile tet Tetr This workRAD6 purAJ6 argA ile tet Tetr This workRAD7 purA16 argA ile tet Tetr This work

Gaithersburg, Md., or New England BioLabs, Inc., Beverly,Mass. Restriction buffers and conditions were as describedby the manufacturer.

Gel electrophoresis. All gels depicted in this paper were 1%agarose with a Trisacetate (40 mM Tris [pH 8.0], 20 mMsodium acetate, 2.5 mM EDTA disodium) electrophoresisbuffer. Gels were electrophoresed at 25 V overnight or at 90V for 4 to 5 h.DNA isolation. In most instances, chromosomal DNA was

prepared from overnight cultures grown in 40-ml volumes ofTSBS with or without Tc (20 ,ug/ml). Cells were pelleted andsuspended in 5 ml of a Tris glucose buffer (50 mM Tris [pH8.0], 50 mM glucose, 10 mM EDTA disodium) containinglysozyme (250 ,ug/ml). After incubation at 37°C for 30 to 40min, 200 ,ul of sodium dodecyl sulfate (10% [wt/vol] in H20)was added to each tube, and the contents were mixedvigorously. The mixture in each tube was transferred to a125-ml Erlenmeyer flask, and 5 ml of a lytic buffer (1% Brij58, 1% deoxycholate, 0.05 M EDTA [pH 7.51) was added.The contents of the flasks were mixed and left at roomtemperature for 10 min before an equal volume of phenol-CHC13 (1:1) was added. All flasks were mixed on a Burrellwrist-action shaker (Burrell Corp., Pittsburgh, Pa.) for 30min and then centrifuged (12,000 rpm for 30 min; DupontSA600 rotor; DuPont Co., Wilmington, Del.). After centri-fugation, the aqueous phase (8 ml) was removed, and theDNA was precipitated by the addition of a 0.1 volume of 3 Msodium acetate and 2 volumes of ethanol. The DNA was

washed three times with 70% ethanol before drying undervacuum. DNA was suspended in 1 ml of TE buffer (10 mMTris [pH 7.5], 1 mM EDTA disodium) and stored at 4°C.DNA prepared by this procedure was suitable for restrictionendonuclease digestion.

Plasmid screen. Tetr isolates were screened for plasmidcontent by agarose gel electrophoresis of (i) total DNAprepared as described above, (ii) minivolume sodium dode-cyl sulfate lysates (22), and (iii) fractions from cesiumchloride-ethidium bromide gradient centrifugation of clearedlysates (22).Mapping experiments with the generalized transducing

phage PBS1. Mapping experiments were performed by a

modification of the procedure of Stan Zahler (unpublishedprocedure; Cornell University). A high titer of PBS1 (109/ml)was obtained by propagation on Bacillus licheniformis.Transducing lysates were prepared by growing donor strainsof B. subtilis to mid-log phase and exposing 1.0-ml volumesof cells to various concentrations of PBS1. Cells and phagewere left at room temperature for 10 min and then incubatedin a shaking incubator at 37°C. After 30 min, chlorampheni-

col was added to a final concentration of 5 ,ug/ml, andincubation was continued for another 2 h. The tubes werethen removed from the shaking incubator and left stationaryat 37°C overnight. The next day, tubes exhibiting lysis werecentrifuged (5,000 x g for 10 min), and the supernatantswere filtered (Gelman Acrodisc disposable filters; 0.45-,umpore size; Gelman Sciences, Inc., Ann Arbor, Mich.). Thetiters of the transducing lysates were checked on B.licheniformis. The strains of B. subtilis used as recipients inmapping experiments are described in Table 1. Mid-log-phase cultures of recipient strains were exposed to transduc-ing lysates, and tubes were aerated for 20 min at 37°C.Control tubes without phage were also subjected to theprocedure. Saline citrate (0.1 M NaCI, 0.05 M sodiumcitrate) (10 ml) was then added, and tubes were centrifugedat room temperature on a Beckman model TJ-6 (2,500 rpmfor 10 min; Beckman Instruments, Inc., Fullerton, Calif.).Cell pellets were suspended in saline citrate before plating onselective medium.

Protoplasting and regeneration. Conditions for theprotoplasting and regeneration of B. subtilis were as de-scribed by Chang and Cohen (7). Selection for Tc resistancewas on DM3 regeneration medium containing 10 or 20 ,ug ofTc per ml.

Inducibility of Tc resistance. Inducibility was tested byincubating one-half of an early-log-phase culture with asubinhibitory concentration of Tc (0.3 ixg/ml). No Tc wasadded to the second half of the culture. After a 70-minincubation period at 37°C, the induced and uninduced por-tions of the culture were used as inocula for lOx volumes ofTSBS with and without 0.3 ,ug of Tc per ml, respectively.After a suitable incubation period, both the induced anduninduced cultures were challenged with an inhibitory finalconcentration of Tc (12.5 ,ug/ml). Incubation at 37°C wascontinued, and growth was monitored by A600 readings.

Stability of Tc resistance. The stability of Tetr was deter-mined by growing Tetr isolates in TSBS in the absence of Tcfor 20 or more generations. Dilutions were plated on nonse-lective medium, and the resulting colonies were screened forTetr. The plating medium (TSAS) was identical to TSBSexcept for the addition of 1.5% agar-agar (Difco Laborato-ries, Detroit, Mich.) and, when appropriate, 20 ,ug of Tc perml.

Level of Tc resistance. Inhibitory concentrations of Tc forboth Tetr and Tets strains of B. subtilis were measured bypatching growth from fresh overnight plates onto mediumcontaining increasing concentrations of Tc. The medium wascomposed of TSAS containing 50 ,ug of guanine per ml. Thefinal Tc concentrations were 2, 10, 20, 50, 100, 150, and 200pg/ml. The last patch of the series was made onto mediumlacking Tc to ensure that sufficient inoculum was transferredthrough the series. Plates were incubated at 37°C for 48 h andobserved for the presence or absence of growth.

RESULTS

Tetr isolates. Tetr strains RAD1, RAD2, RAD6, and RAD7were isolated after the protoplasting and regeneration of B.subtilis BC92. Initially, the occurrence of Tetr appeared tobe dependent on transformation with the plasmid pCW59,which encodes resistance to chloramphenicol and Tc (22).Subsequent experiments demonstrated that pCW59 DNAwas not required and that Tetr isolates occurred at a lowfrequency (<10-8) after protoplasts of B. subtilis weretreated with polyethylene glycol and regenerated on DM3agar containing 20 ,ug of Tc per ml. Approximately 90% of

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DNA AMPLIFICATION IN B. SUBTILIS 447

the initial cells formed protoplasts, and 30% of these wereregenerated on DM3.No Tetr were observed in control experiments in which

nonprotoplasted cells were plated directly onto TSAS andDM3 media containing Tc.Two experiments were devised to determine whether

amplified DNA was a common occurrence in cells whichsurvived protoplasting, polyethylene glycol fusion, and re-generation. In the first experiment, protoplasts treated withpolyethylene glycol were plated on regeneration mediumwithout antibiotic. In the second experiment, regeneratedprotoplasts transformed by the Kmr plasmid pUB110 wereselected. In both experiments, 12 colonies were chosen atrandom, and total DNA was isolated as described above.Restriction digests of these DNAs did not reveal the pres-ence of amplified DNA bands other than the pUB110 bandpresent in DNA from the Kmr transformants.

Screen for plasmid DNA. All Tetr isolates were extensivelyscreened for the presence of plasmid DNA. Cleared lysatesof the Tetr isolates and control strains of B. subtilis were

E

i0

a

0

A

0. *C92o

a a A I I

TIME (hr)

FIG. 1. Testing the inducibility of Tc resistance. One-half ofearly-log-phase cultures of RAD1, RAD2, and BC92 were incubatedwith a subinhibitory concentration of Tc (0.3 p.g/ml). After incuba-tion at 37°C for 70 min., both induced and uninduced portions of theculture were used to inoculate lOx volumes of broth with andwithout Tc (0.3 p.g/ml), respectively. Incubation at 37°C was con-

tinued for an additional 60 to 90 min., and then both the induced anduninduced cultures were challenged (arrows) with an inhibitory finalconcentration of Tc (12.5 ,ug/ml). Growth was monitored by A6wreadings. 0, No induction; 0, induced.

TABLE 2. Growth' of Tets and Tetr strains of B. subtilis atdifferent concentrations of Tc

Tc concn (pug/ml)Strain

2 10 20 50 100 150 200

RAD1 + + + + + + -RAD2 + + + + + + -RAD6 + + + + + - -RAD7 + + + + + - -BC92(pCW59) + + + + + + +BC92 + - - - - - -

IA177 + - - - - - -QB944 + - _ _ _ _ _

a +, Growth; -, no growth.

compared by agarose gel electrophoresis. The lysates wereprepared either by a sodium dodecyl sulfate-cleared lysateprocedure described previously (22) or by a modifiedBirnboim and Doly (6) procedure. No plasmid DNA wasdetected in either the Tetr isolates or any of the recipientstrains of B. subtilis used in the transduction mappingexperiments (data not shown).The total DNA prepared from the Tetr isolates and from

BC92 by the Brij 58-deoxycholate procedure was subjectedto cesium chloride-ethidium bromide density gradient cen-trifugation (see above). After achieving equilibrium, thegradients were fractionated, and the fractions were exam-ined by agarose gel electrophoresis. No plasmid DNA wasdetected for any Tetr isolate or for the parental BC92 strain(data not shown).

Inducibility of Tc resistance. The inducible or constitutivenature of the Tetr determinants present in RAD1 and RAD2was examined (Fig. 1). The experimental protocol is de-scribed above and in the legend to Fig. 1. Early in log phase,uninduced and induced (subinhibitory concentration of Tc)cultures were challenged with Tc at a final concentration of12.5 ,ug/ml. Growth was monitored by Ao00 readings. Growthof both the induced and uninduced cultures of BC92 wasinhibited by the challenge dose of Tc. Inhibition of growthalso occurred for the uninduced cultures ofRAD1 and RAD2(Fig. 1, open circles). In contrast, no significant inhibition ofgrowth was observed for the induced cultures of RAD1 andRAD2 (Fig. 1, closed circles). The inhibition of theuninduced cultures was transient. By 6 h after challenge withthe higher concentration of Tc, the A6N readings for theuninduced culture of RAD1 started to increase. This re-covery was also observed in the other Tetr isolates (data notshown). After overnight growth, the A6w readings for theuninduced cultures were, in most instances, equal to orgreater than the highest A6w reading observed for thecorresponding induced cultures.

Level of Tetr. The levels of Tetr in the strains listed inTable 2 were determined by the procedure described above.All of the Tetr strains derived from BC92 grew at Tcconcentrations up to 100 ,ug/ml. The parental BC92 strainand strains IA177 and QB944, which were used as recipientsin transductional crosses, were inhibited by 10 ,ug of Tc perml. Differences in resistance levels between RAD1, RAD2,RAD6, and RAD7 were shown by the inability of the lattertwo isolates to grow at 150 p.g of Tc per ml. BC92 carryingCmr Tcr plasmid pCW59 was able to grow at the highest Tcconcentration (200 ,ug/ml) tested.

Stability of Tetr. The stability of Tetr associated withisolates RAD1, RAD2, RAD6, and RAD7 was measured asdescribed above. Dilutions from the second and third passes

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448 WILSON AND MORGAN

TABLE 3. Stability of Tc resistance"

Growth (no. of % ColoniesB. subtilis generations) in resistant to

strain the absence of TcTc

RAD1 .20 97.30 97

RAD2 .20 100.30 100

RAD6 .20 97.30 97

RAD7 .20 82.30 71

BC92(pCW59) .20 9

a The experimental protocol is described in the text.

of growth in nonselective broth were plated on TSAS plateswithout Tc. Colonies were picked from these plates ontoTSAS plates containing 20 ,ug of Tc per ml. At least 100colonies from each isolate were screened for Tetr (Table 3).Tc resistance associated with RAD1, RAD2, and RAD6 wasvery stable. After 30 generations of growth in the absence ofselection, 97% of the colonies from RAD1 and RAD6 and100% of the colonies from RAD2 retained resistance to Tc.The resistance determinant associated with RAD7 was lessstable with only 71% of the colonies resistant to Tc. As acomparison, B. subtilis BC92 carrying plasmid pCW59 (CmrTcr) was carried through a similar stability study. After 25generations of growth in the absence of selection, only 9% ofthe colonies were still resistant to Tc.

Amplified regions of chromosomal DNA present in Tetrisolates. Chromosomal DNAs prepared from several Tetrisolates and from BC92 were compared by agarose gelelectrophoresis of HindIll and EcoRI restriction digests(Fig. 2 and 3). Surprisingly, digests of DNA from Tetrisolates RAD1, RAD6, and RAD7 contained restrictionfragments which appeared amplified when compared withidentical digests of BC92 DNA. No amplified fragments wereobserved in HindlIl, EcoRI, BglII, or ClaI digests of RAD2DNA. The amplified fragments present in the HindIll andEcoRI digests of DNA from RAD1, RAD6, and RAD7differed in size and number. The two amplified fragmentsvisible in the HindlIl digest of RAD1 DNA were 4.9 and 4.5kilobases in length. The amplified fragments visible inHindIII digests of RAD6 and RAD7 DNA were larger andsmaller than those of RADI DNA and differed from eachother. HindlIl digests of plasmids pCW59 (22) and pLS11(17) are included in Fig. 2 for comparison.Mapping Tc resistance determinants. The generalized

transducing phage PBS1 was used to map resistance to Tc(tet) on the B. subtilis chromosome. Transduction protocolsare described above. The donor and recipient strains are

described in Tables 1 and 4. The mapping results arecontained in Table 4 and summarized in Table 5. PBS1transducing lysates prepared from RAD1 and RAD6 showedsimilar but different cotransfer frequencies between tet andthe purA, guaA, and cysA loci. The cotransfer frequenciesfor tet from RAD1 and RAD6 were quite different from thoseobserved for tet from RAD2. In all three isolates, tet mappedclose to the guaA locus. This was particularly true forRAD2, in which 84% of the Gua+ transductants were resist-

ant to Tc. In all three isolates, linkage was also evidentbetween tet and the purA locus and, to a lesser extent, thecysA locus. Again, tet from RAD2 showed the highestcotransfer (62%) with purA. The genotype of BC92 fromwhich RADI, RAD2, and RAD6 were derived complicatedcrosses involving purA. For example, recipient strain QB944also carried the purA16 mutation. Linkage of tet with purAwas scored in transduction experiments with strain IA177 as

the recipient (Table 4). Linkage was measured by scoringGua+ Tetr transductants which were Pur-. Results from thetransduction studies indicate that in all three isolates, tetmaps close to guaA and very near to the origin of replicationof the B. suibtilis chromosome (12, 23; D. Ziegler and D.Dean, FEMS Microbiol. Rev., in press). The mapping datawere not sufficient to orient tet in RADI, RAD2, or RAD6clockwise or counterclockwise to guaA. The frequencies ofcotransfer (Table 5) show the nonreciprocal nature of thetransductional crosses when initial selection was for Tetr. Inevery case, initial selection for Tetr gave fewer transduc-tants. These results indicate that cotransfer frequenciescalculated from initial selection for Tetr should, therefore, beviewed cautiously.

X0N

Hind III digests

> > > > r

aa

%J (0a 14M I

FIG. 2. Agarose gel electrophoresis of Hindlll digests of chro-mosomal DNA prepared from BC92 and the Tetr strains derivedfrom BC92. Amplified fragments (0) are visible in digests of DNAfrom RAD1, RAD6, and RAD7 but not in digests of DNA fromRAD2 and BC92. Hindlll digests of pCW59 (23) and pLS11 (16)plasmid DNAs are included for comparison.

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DNA AMPLIFICATION IN B. SUBTILIS 449

Cotransduction of amplified DNA with Tetr. Initial mappingresults 'from PBS1 prop'agated on RAD1, RAD2, and RAD6indicated linkage between Tc resistance and the cysA locus(Table 4). Restriction digests of chromosomal DNA pre-pared from QB944 transduced to Cys+ Tetr by PBS1 propa-gated on RAD1, RAD2, and RAD6 were compared byagarose gel electrophoresis (Fig. 4-6). The amplified DNAfragments characteristic of HindIII digests of RAD1 andRAD6 DNA were present in digests of chromosomal DNAfrom the corresponding Cys+ Tetr transductants of QB944.No amplified bands were present in HindIII digests of eitherthe parental RAD2 DNA or DNA from the correspondingCys+ Tetr transductants of QB944. The possibility thatamplified DNA might be cotransduced with the cysA locusalone was examined in the same manner. A total of 30 Cys+Tets transductants of QB944 were screened for the presenceof amplified DNA fragments in restriction digests. The Cys'Tets isolates were equally distributed between QB944transduced with PBS1 propagated on RAD1 and RAD6. Noamplified-DNA fragments were observed in HindIlI digestsof chromosomal DNA from any of the Cys+ Tet5 isolates(Fig. 5 and 6).

Subsequent mapping results from transduction experi-ments with IA177 as the recipient showed a higher frequencyof cotransfer of Tetr with Gua+ than with Cys+ (Table 5).Again, the amplified DNA characteristic ofRAD1 and RAD6was present in HindIII digests of chromosomal DNA fromGua+ Tetr (Fig. 7) and Gua- Tetr transductants (data notshown). No amplified fragments were present in HindIIIdigests of chromosomal DNA from Gua+ or Gua+ Pur-transductants (Fig. 7).

Loss of Tetr accompanied by loss of amplified DNA. Theamplified DNA present in RAD1 and 'RAD6 wascotransduced with Tetr to recipient QB944 (Fig. 5 and 6).This close association between Tetr and amplified DNA wasfurther characterized by comparing restriction digests ofchromosomal DNA prepared from one Tetr and three Tetsrevertants of RAD1, RAD6, and RAD7 (Fig. 8). In all cases,reversion to Tets was accompanied by loss of amplified-DNA fragments from restriction digests.

DISCUSSIONIn this paper, we describe four Tetr isolates of B. subtilis

BC92. Tetr strains RAD1, RAD2, RAD6, and RAD7 wereisolated after the protoplasting of BC92, polyethylene glycoltreatment, and regeneration on medium containing Tc. Allfour isolates grew well at a Tc concentration of 100 ,ug/ml,but only RAD1 and RAD2 grew at a concentration of 150,ug/ml. Resistance to Tc was inducible by subinhibitoryconcentrations of the antibiotic (Fig. 1). The inducible natureof the Tetr phenotype associated with RAD1 and RAD2 wascommon to all four isolates (data not shown).'The characteristic that distinguishes these Tetr strains

from those previously reported in the literature is the pres-ence of amplified chromosomal DNA, which is closelyassociated with Tc resistance. Restriction digests of chro-mosomal DNA from RAD1, RAD6, and RAD7 containedamplified fragments (Fig. 2 and 3). The intensity of thefragments indicates the presence of tandemly duplicatedDNA within the chromosome. The HindIII and EcoRIdigests did not reveal any amplified fragments common to allthree isolates. Disparity in the size and number of amplifiedfragments suggests that the tandemly duplicated DNA isdifferent in all three isolates. The size of the tandemlyduplicated DNA present in each isolate can be estimated bysummation of the sizes of the amplified fragments present in

Eco RI digests

> > > > _0 a 0 aX (D :-& Ml 4) -J hi Q

23.1

9.4

6.7

.0

4.4 _ewNu,

2.3

2.0

FIG. 3. Agarose gel electrophoresis of EcoRI digests of chromo-somal DNA prepared from BC92 and the Tetr strains derived fromBC92. Amplified fragments (0) are visible in digests of DNA fromRAD1, RAD6, and RAD7 but not in digests ofDNA from RAD2 andBC92. A HindlIl digest of lambda is included for a molecular-sizereference. kb, Kilobase.

restriction digests. Based on a limited number of digests, theestimated sizes of the duplicated DNAs present in RAD1,RAD6, and RAD7 were 10, 19, and 20 kilobases, respec-tively.The possibility that the amplified DNA present in RAD1,

RAD6, and RAD7 might reflect amplified synthesis of a newor altered protein was examined. Total protein preparationsfrom all four Tetr isolates and BC92 were compared bysodium dodecyl sulfate-urea acrylamide gel electrophoresis.No differences in the intensity or pattern of protein bandswere detected between any of the Tetr isolates and BC92(data not shown).No amplified fragments were present in digests of RAD2

chromosomal DNA. The resistance of RAD2 to Tc wasequal to that of RAD1 and greater than that of RAD6 andRAD7 (Table 2). This implies that DNA amplification is notrequired for the high level of Tc resistance observed in thefour isolates. However, results of mapping experiments withthe generalized transducing phage PBS1 indicate a closeassociation between amplified DNA and Tetr in strainsRAD1 and RAD6. Amplified-DNA fragments characteristicof HindIII-digested RAD1 and RAD6 DNA were present in

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450 WILSON AND MORGAN

TABLE 4. Mapping tet by PBS1 transduction"Selected Markerf

B. subtilis recipient strain (genotype) marker and No. No. of(RDnr tested cysA rrpC arol tet purA guaA recombinants

QB944 (parAJ6 cysA14 trpC2) cysA+1

2

6

trpC+1

2

6

tet1

2

6

256 1 01 0

440 1 01 0

768 1 01 0

200 0 1

209 0 1

240 0 1

50 0 01 0

87 1 00 0

29 0 0

10

10

10

23233

143297

51717

0

0

200

209

0

11

11

1

240

464

879

29

QB928 (aroI906 purB33 dal-l trpC2) aroI+1 5 0 1 0

0 1 0

0 1 0

IA177 (guaAl rpIV) guaA11 1 11 0 10 1 10 0 1

1 1 11 0 10 1 10 0 1

1 1 11 0 10 1 10 0 1

1 1 01 0 0

tet (RAD1) 37

a tet encodes resistance to Tc.b Donor genotypes are described in Table 1.Donor, 1; recipient, 0.

digests of DNA isolated from the corresponding Tetr trans-ductants of QB944 and IA177 (Fig. 4-7). Attempts to dem-onstrate the transduction of amplified DNA with the nearbyloci guaA+ and cysA+ in the absence of Tetr were notsuccessful (Fig. 5-7).Loss of Tetr by RAD1, RAD6, and RAD7 was ac-

companied by loss of amplified DNA. All three strains weregrown for over 20 generations in the absence of Tc beforeplating on nonselective medium. Restriction digests of chro-mosomal DNA prepared from Tetr and Tets colonies of

RAD1, RAD6, and RAD7 were compared by agarose gelelectrophoresis. The amplified fragments characteristic ofthe three Tetr isolates were not present in the HindlIl digestsof DNA from the corresponding Tet' revertants (Fig. 8).Most of the chromosomal tet mutations in B. subtilis

which have been characterized map within the principalribosomal-protein-gene cluster and involve the alteration ofone or more ribosomal proteins (8-10, 21). In B. subtilis, thisgene cluster maps near the origin of chromosomal replicationand clockwise to the cysA locus (9, 10). Results of

2

6

45

480

5

45

480

2

6

360

360

160139

9231

223793

55

549810

198

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DNA AMPLIFICATION IN B. SUBTILIS 451

TABLE 5. Summary of cotransfer data from transductionexperiments with phage PBS1

Cotransfer (%)bDonor'

tet purA 16 tet guaA+ tet cysA+ tet aroIl

RAD1 30c, (12)b 55, (0) 9, (8) 0RAD2 62 84 33, (9) 0RAD6 15 42 7 0

a Donor strains are described in Table 1.b tet encodes resistance to Tc.c Initial selection was for guaA+. This percentage was determined from the

number of guaA+ Tetr transductants which also received the donor purAlocus.

d Numbers in parentheses represent cotransfer (%) when initial selectionwas for tet.

transductional-mapping studies with PBS1 showed that thetet loci of RAD1, RAD2, and RAD6 also map near the originor replication of the B. subtilis chromosome and close to theguaA locus (Table 5). In all three strains, tet also mappednear the purA and cysA loci. The order of the three loci onthe B. subtilis chromosome is purA, guaA, and cysA (19;Ziegler and Dean, in press). The purA locus lies counter-clockwise, and the guaA and cysA loci lie clockwise to the

cm

41t

aa

CaS.

Cyst TcIt ra usductants

PBS1 propagated on B.S. RADI

Cyst TcR .Wi.P trassductaits .CA

X PBSI from -

2 B.S. RAD21

C 1s TcRtransductants

PBSI fron B.S. RAMI

FIG. 4. Agarose gel electrophoresis of HindIII digests of chro-mosomal DNA prepared from RAD1, RAD2, BC92, QB944, andTetr transductants of QB944. Chromosomal DNA was isolated fromQB944 transduced to Tetr with PBS1 propagated on either RADM orRAD2. Initial selection was for Tetr, but all of the transductantsdepicted in this gel also received the cys+ locus from the donorstrain. The amplified fragments characteristic of HindlIl digests ofRADM DNA were present in digests ofDNA from QB944 transducedby PBS1 propagated on RADL. As expected, no amplified fragmentswere visible in digests of DNA from BC92, QB944, and RAD2 or indigests of DNA from QB944 transduced by PBS1 propagated onRAD2. B. S., B. subtilis; TCR, Tetr.

FIG. 5. Agarose gel electrophoresis of Hindlll digests of chro-mosomal DNA prepared from strain QB944 transduced to Cys+ orCys+ Tetr with PBS1 propagated on RADL. Initial selection was forCyst. Amplified fragments characteristic of Hindlll digests ofRADM DNA were only present in digests of DNA isolated from Tetrtransductants. Digests of 3 of 15 DNA samples isolated from Cys+TetV transductants screened for the presence of amplified fragmentsare included in the gel. B. S., B. subtilis; TcR, Tetr; Tcs, Tets.

origin of chromosomal replication. The degree of linkagebetween tet and purA, guaA, and cysA varied for eachisolate and was affected by the marker initially selected aftertransduction (Table 5). The tet locus of RAD2 showed ahigher frequency of cotransfer with the guaA and purA locithan did the tet loci of RADM and RAD6. The increasedlinkage between tet and guaA and purA in RAD2 may resultfrom the absence of amplified DNA in that isolate. Thepresence of tandemly duplicated DNA in RADM and RAD6might be expected to alter linkage between tet and surround-ing loci if those loci were not included within the tandemlyrepeated unit. In all three isolates, the tet locus was moreclosely linked to guaA and purA than to cysA (Table 5).These results positioned tet counterclockwise to cysA anddistant from the principal ribosomal-protein-gene cluster onthe B. subtilis chromosome. Williams and Smith (21) havealso described a tet locus which maps in this region of the B.subtilis chromosome. Interestingly, the region of the B.subtilis chromosome between guaA and cysA contains anrRNA gene set (rrnA) consisting of genes for 16S, 23S, 5S,and 4S rRNAs (23). The rrnA locus maps closer to guaA thanto cysA and contains 7 to 10 copies of the rRNA genesseparated by nonhomologous DNA spacers.

Cys+ TcSt ransductas ts

co qW2 !f:0 w:b CICDa_. l

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452 WILSON AND MORGAN

cy+ TK Cys4 TeCSCys tcraGsdtstrausductants tranisductants

Ia+T u+cTsu4 Tra r + Tc11 ir|_Pllte | e a TsA P r

W itdalsictsllts of IA1Z77 wi tk PISI propagated on1 1laolPBSI propagated on B.S. RAD6

FIG. 7. Agarose gel electrophoresis of HindIll digests of chro-mosomal DNA prepared from strain IA177 transduced to Gua+ withPBS1 propagated on RADL. Amplified fragments characteristic ofHindlll digests of RADi DNA were only present in Gua+ transduc-tants which also received Tetr. Transductants which received thepur- locus, but not Tetr, from RADM did not contain amplified DNA.TcR, Tetr; Tcs, Tets.

FIG. 6. Agarose gel electrophoresis of Hindlll digests of chro-mosomal DNA prepared from strain QB944 transduced to Cys+ or

Cys+ Tetr with PBS1 propagated on RAD6. Initial selection was forCyst. Amplified fragments characteristic of HindIll digests ofRAD6 DNA were only present in digests of DNA isolated from Tetrtransductants. No amplified fragments were observed in HindIlldigests of 15 DNA samples prepared from Cys+ Tet5 transductants.Of the 15 digests, 3 are included in the gel. B. S., B. subtilis; TCR,Tetr; TcS, Tet5.

In Escherichia coli and Salmonella typhimurium, tandemduplications of chromosomal DNA have occurred afterrecombination between rRNA genes (5, 13). As much as 3%of a Salmonella population was reported to have spontane-ous duplications which arose from unequal crossing overbetween dispersed repeated sequences of rRNA cistrons (5).Most of these duplications were near the origin of chromo-somal replication. In E. coli, duplications of the gly+ purAchromosomal region resulted from uneven crossing overbetween rRNA genes on either side of the loci (13).A plausible explanation of the amplified DNA present in

RAD1, RAD6, and RAD7 would be the occurrence ofchromosomal duplications as a result of unequal recombina-tion between rRNA cistrons in the region between guaA andcysA. This recombination could occur between differentchromosomes or between daughter chromatids present in areplication fork. The proximity of the guaA and cysA loci tothe chromosomal bidirectional origin or replication makesthe latter possibility more likely. The presence of genesencoding ribosomal proteins in the guaA-to-cysA region also

B.S. RAD6

Tc_T== Ts C

B.S. RlAp

T5 TeS TcaITcSI.$. NADl

TcS TcS TcR

FIG. 8. Loss of Tet accompanied by loss of amplified DNA.After more than 20 generations of growth in the absence of Tc,chromosomal DNA was prepared from one Tet and three Tetscolonies of RADi, RAD6, and RAD7. HindIll digests of the DNAwere compared by electrophoresis on a 1% agarose gel. In eachcase, amplified DNA fragments were not visible in digests of DNAprepared from TetV revertants. B. S., B. subtilis; Tcs, Tet8; TcRTetr.

r.. ......... ......... .... - -= --

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DNA AMPLIFICATION IN B. SUBTILIS 453

makes it likely that the tet locus in all four isolates lies closeto the duplicated DNA region.The high-level, inducible resistance to Tc which character-

izes the RAD mutants would be more easily explained by thepresence of a Tcr plasmid. However, by using three differentexperimental approaches, we were unable to detect plasmidDNA in any of the RAD mutants. In addition, the transduc-tion mapping data clearly demonstrated the chromosomallocation of Tc resistance in the three RAD mutants tested.Chromosomal mutations producing resistance to Tc gener-ally result from the alteration of ribosomal proteins (8, 21).The Tc resistance associated with these ribosomal mutationsis neither inducible nor of a high level. Recently, Shishido etal. (16) reported that B. subtilis carries an integrated plasmidsequence which encodes resistance to Tc. When integratedin the chromosome, the Tcr determinant is under negativeregulatory control. Expression only occurs after excisionand autonomous replication as a plasmid. Unfortunately, noinformation has been published concerning the location ofthis Tc resistance determinant on the chromosomal-DNAmap of B. subtilis.

ACKNOWLEDGMENTSWe are grateful to Yvonne Frazer for technical assistance, Steve

Phillips for helpful discussions, Ernie Navarro for photographicassistance, and Nancy Coppler for preparation of the manuscript.

LITERATURE CITED

1. Alt, F. W., R. E. Kellems, J. R. Bertno, and R. T. Schimke.1978. Selective multiplication of dihydrofolate reductase genesin methotrexate-resistant variants of cultured murine cells. J.Biol. Chem. 253:1357-1370.

2.A, C., and J. Spizizen. 1%1. Requirements fortransformation in Bacillus subtilis. J. Bacteriol. 81:741-746.

3. Anderson, R. P., C. G. Miler, and J. R. Roth. 1976. Tandemduplications of the histidine operon observed following general-ized transduction in Salmonella typhimurium. J. Mol. Biol.105:201-218.

4. Anderson, R. P., and J. R. Roth. 1977. Tandem genetic duplica-tions in phage and bacteria. Annu. Rev. Microbiol. 31:473-505.

5. Anderson, R. P., and J. R. Roth. 1981. Spontaneous tandemgenetic duplications in Salmonella typhimurium arise by un-equal recombination between rRNA (rrn) cistrons. Proc. Natl.Acad. Sci. U.S.A. 78:3113-3117.

6. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline procedurefor screening recombinant plasmid DNA. Nucleic Acids Res.7:1513-1523.

7. Chang, S., and S. N. Cohen. 1979. High frequency transforma-tion of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen.Genet. 168:111-115.

8. Dabbs, E. R. 1982. Selection in Bacillus subtilis giving rise tostrains with mutational alterations in a variety of ribosomalproteins. Mol. Gen. Genet. 187:297-301.

9. Dabbs, E. R. 1983. Arrangement of loci within the principalcluster of ribosomal protein genes of Bacillus subtilis. Mol. Gen.Genet. 192:124-130.

10. Dabbs. E. R. 1984. Order of ribosomal protein genes in the Rifcluster of Bacillus subtilis is identical to that of Escherichia coli.J. Bacteriol. 159:770-772.

11. Fishman, S. E., and C. L. Hershberger. 1983. Amplified DNA inStreptomyces fradiae. J. Bacteriol. 155:459-466.

12. Haldenwang, W. G., C. D. B. Banner, J. F. OUington, R. Losick,J. A. Hoch, M. B. O'Connor, and A. L. Sonenshein. 1980.Mapping a cloned gene under sporulation control by insertion ofa drug resistance marker into the Bacillus subtilis chromosome.J. Bacteriol. 142:90-98.

13. Hill, C. W., R. H. Grafstrom, B. W. Harnish, and B. S. Hilhman.1977. Tandem duplications resulting from recombination be-tween ribosomal RNA genes in Escherichia coli. J. Mol. Biol.116:407-428.

14. Nunberg, J. H., R. J. Kaufman, R. T. Schimke, G. Urlaub, andL. A. Chasin. 1978. Amplified dihydrofolate reductase genes arelocalized to a homogeneously staining region of a single chromo-some in a methotrexate-resistant Chinese hamster cell line.Proc. Natl. Acad. Sci. U.S.A. 75:5553-5556.

15. Saito, H., H. Anzai, and F. Kawamura. 1983. Multicopy integra-tion vectors in Bacillus subtilis; p. 125-130. In Y. Ikeda and T.Beppu (ed.), Proceedings of the Fourth International Sympo-sium on Genetics of Industrial Microorganisms. Kodansha Ltd.,Tokyo, Japan.

16. Shishido, K., N. Noguchi, C. Kim, and T. Ando. 1983. Isolationof a tetracycline-resistance plasmid excised from a chromo-somal DNA sequence in Bacillus subtilis. Plasmid 10:224-234.

17. Tanaka, T., M. Kuroda, and K. Sakaguchi. 1977. Isolation andcharacterization of four plasmids from Bacillus subtilis. J.Bacteriol. 129:1487-1494.

18. Tisty, T. D., A. M. Albertini, and J. H. Miller. 1984. Geneamplification in the lac region of E. coli. Cell 37:217-224.

19. Trowsdale, J., S. M. H. Chen, and J. A. Hoch. 1979. Geneticanalysis of a class of polymyxin resistant partial revertants ofstage 0 sporulation mutants of Bacillus subtilis: map of thechromosome region near the origin of replication. Mol. Gen.Genet. 173:61-70.

20. Vasseghi, H., and J.-P. Claverys. 1983. Amplification of achimeric plasmid carrying an erythromycin-resistance determi-nant introduced into the genome of Streptococcus pneumoniae.Gene 21:285-292.

21. Williams, G., and I. Smith. 1979. Chromosomal mutationscausing resistance to tetracycline in Bacillus subtilis. Mol. Gen.Genet. 177:23-29.

22. Wilson, C. R., S. Skinner, and W. V. Shaw. 1981. Analysis oftwo chloramphenicol resistance plasmids from Staphylococcusaureus: insertional inactivation of Cm resistance, mapping ofrestriction sites and construction of cloning vehicles. Plasmid5:245-258.

23. Wilson, F. E., J. A. Hoch, and K. Bott. 1981. Genetic mappingof a linked cluster of ribosomal ribonucleic acid genes inBacillus subtilis. J. Bacteriol. 148:624-628.

24. Young, M. 1984. Gene amplification in Bacillus subtilis. J. Gen.Microbiol. 130:1613-1621.

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