Vol. 173, No. 19

Heterozygosity and Instability of Amplified Chromosomal Insertionsin the Radioresistant Bacterium Deinococcus radioduransC. IAN MASTERS, MICHAEL D. SMITH,t PABLO D. GUTMAN, AND KENNETH W. MINTON*Department ofPathology, F. Edward Hebert School of Medicine, Uniformed Services University of the

Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 208144799

Received 18 April 1991/Accepted 24 July 1991

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, abacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were oftenheterozygous, with some chromosomes containing highly amplified insertions and others containing no

insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as

by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. Thecircles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strainsthat are defective in natural transformation and sensitive to DNA-damaging agents were further characterized.Both strains were defective in duplication insertion. While one strain was normal for plasmid transformation,the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may requirerecombinational functions.

Bacteria of the genus Deinococcus share an extremedegree of resistance to the lethal and mutagenic effects ofionizing and UV radiation and to most other agents thatdamage DNA (9). The most studied of these species, Deino-coccus radiodurans, was found to be naturally transformableby Moseley and coworkers, who showed that this organismis always competent during the exponential phase withoutmanipulation and provided evidence, by using unlinkedmarkers, for a minimum of four independently segregatinggenomes per cell (13, 21). The latter observation is consis-tent with evidence based on renaturation kinetics (5) andpulsed-field gel electrophoresis (4) that D. radiodurans con-

tains 4 chromosomes per nucleoid in the plateau phase and 8to 10 chromosomes during exponential growth (9). Very littleis known about transformation in D. radiodurans, in contrastto a number of relatively well-characterized naturally trans-formable organisms, such as Haemophilus, Bacillus, or

Streptococcus spp. (2, 8, 15).We have lately reported expression of heterologous drug

resistance determinants in D. radiodurans, permitting chro-mosomal duplication insertions (7, 17) and the constructionof autonomously replicating vectors that express these de-terminants (16, 18). Exploiting these capabilities, we haveused duplication insertion, plasmid transformation, and nat-ural transformation to further characterize transformationand recombination properties of wild-type (wt) D. radiodu-rans, as well as two strains previously shown to be defectivein natural transformation, rec30 (10) and 112 (3, 4). Thesestudies demonstrate that D. radiodurans possesses a highlyproficient recombination ability, including efficient duplica-tion insertion, gene amplification, and frequent chromo-somal deletions between distant homologous regions of largeamplifications.

* Corresponding author.t Present address: Life Technologies, Inc., Gaithersburg, MD

20877.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Bacte-rial strains, plasmids, and growth conditions are described inTable 1.

Transformation. Exponential cultures of D. radioduranswere resuspended at 109 cells per ml in TGY broth (0.8%Bacto-Tryptone, 0.1% glucose, 0.4% Bacto-Yeast; DifcoLaboratories)-0.1 M CaCl2glycerol (20/8/3, vol/vol/vol),frozen on powdered dry ice, and stored at -70°C untilneeded. For transformation, 100 ,ul of the suspension wasthawed on ice (4°C) and no more than 10 ,il of watercontaining various amounts of transforming DNA was

added. The cell mixture was held on ice for 15 min and thenincubated at 32°C for 45 min with gentle agitation. TGY (3 to5 ml) was then added, and the mixture was incubated at 32°Cfor 16 h with aeration, prior to being plated on drug-selectiveagar as noted in Table 1.DNA isolation and manipulation. Isolation of genomic

DNA and purified plasmid from D. radiodurans and Esche-richia coli and use of enzymatic reagents, gel electrophore-sis, blotting, hybridization, washing of blots, and autoradi-ography were as previously described (7). All DNAfragments employed as probes for hybridizations were elec-troeluted from the agarose gel prior to nick translation.

Survival measurements. Exponential-phase cultures of 1.0x 108 cells per ml were employed. Challenges with 'Coirradiation, UV (254 nm), and mitomycin C (MMC) were aspreviously described (4). Samples were taken at intervals,diluted, plated on TGY agar, and allowed to grow at 32°C for2 to 3 days prior to the counting of colonies.

RESULTSTransformation of wt and strains rec30 and 112. At high

concentrations of genomic DNA from the Rifr strain R1-R,transformation of wt to Rif' approached 1%, while transfor-mation of rec30 to Rif' was 100-fold less, and transformationof strain 112 was not detectable (Fig. 1A). Strain rec30,which is unusually sensitive to MMC (Fig. 2), was trans-formed to MMC resistance by genomic DNA from wt (Fig.1A) or strain 112 (not shown) at the same frequency as

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TABLE 1. Bacterial strains and plasmids

Species and strain Source and/oror plasmid Description' referenceb

StrainsD. radioduranscRl wt; MMC and UV resistant 9R1-R Rif; spontaneous mutation This work; Rl x Rl Krase (21)rec30 MMC and UV sensitive MNNG-mutagenized derivative of wt Ri (10)112 MMC and UV sensitive; mtcA 3; also referred to as strain recl; MNNG-

mutagenized derivative of mtcA strain 30230/wt MMC and UV resistant This study; rec30 x Rl30/1 MMC and UV resistant This study; rec30 x 112R1/Tn9 RlflpUC19::Tn9; Cmr This study; Rl x [pUC19::Tn9] HindIII::R1

HindIII

E. coli DH5a recAl endAI hsdRJ7 (rK- MK-) F- 80d Bethesda Research LaboratorieslacZ/M15

PlasmidsdpI3 E. coli-D. radiodurans shuttle plasmid; This study; pS30 (18) derivative in which

Cmr in D. radiodurans; Apr in E. coli multicloning site is moved from Cmr geneto near Apr gene

pELl Cmr; P15A replicon; 3.2 kb 17pEL2 pELl BamHI-BclI-A::12-kb Ri 17

chromosomal BclI fragmentpPG80e pELl BamHI::pEL2 EcoRI-B This study; blunt-end ligationpPG81e pELl BamHI::pEL2 EcoRI-B This study; blunt-end ligationpPG83 pELl BamHI::pEL2 EcoRI-A This study; blunt-end ligationa Ap, ampicillin; Cm, chloramphenicol; Rif, rifampin; r, drug resistance; ::, linkage; Qi, chromosomal insertion.b Transformations are designated recipient strain x donor DNA. MNNG, N-methyl-N'-nitro-N-nitrosoguanidine.c D. radiodurans strains were grown at 32°C in TGY broth or on TGY plates solidified with 1.5% agar. Selective drug concentrations in TGY plates were 25

,ug of rifampin, 3 ,ug of chloramphenicol, or 0.003 ,g of MMC per ml.d Plasmids are described in terms of parental restriction fragment as previously described (7, 16-18). For example, the description of pPG80 as pELl

BamHI::pEL2 EcoRI-B indicates that pELl, cleaved at its solitary BamHI site, is joined to the second largest (i.e., B) EcoRI fragment of pEL2.e pPG80 and pPG81 are identical to each other, except for the opposite orientation of pELl.

transformation of rec30 to Rif' (Fig. 1A). Since the Rif' pointmarker used is the most efficiently transformed markerdiscovered to date (21), the equally high level of efficiency oftransformation to MMC resistance by using either wt orstrain 112 genomic DNA suggests that the rec30 defect is dueto a single mutation and, furthermore, that the rec30 muta-tion is not closely linked with the 112 defect. Two rec30MMC-resistant isolates, one transformed by wt genomicDNA (30/wt) and one transformed by strain 112 genomicDNA (30/1), were found to be wt in all respects, includingefficient natural transformation (Fig. 1A), duplication inser-tion (Fig. 1B), and restoration of normal resistance toDNA-damaging agents (Fig. 2), further supporting the con-clusion that the rec30 phenotype can be assigned to a singlegene. Duplication insertion, measured by using pEL2 (Cmr)(see Fig. 4A), at high DNA concentrations yielded transfor-mation frequencies approaching 0.1% in wt, while rec30 wastransformed to Cmr at a frequency 2,000-fold less, andinsertion in strain 112 was not detectable (Fig. 1B). Unlikewt and rec30, strain 112 was defective in plasmid transfer(Fig. 1C).Uptake of DNA. Because strain 112 was globally defective

in transformation, including plasmid transfer, we evaluateduptake of DNA (Table 2). Strain rec30 showed uptake oflinear and circular DNA into a DNase I-resistant state thatwas similar to wt uptake. Strain 112, however, showed areproducible difference. It bound more linear or circularDNA (prior to DNase I treatment), and it showed enhanceduptake of linear and circular DNA achieving a DNaseI-resistant state.

Duplication insertions are heterozygous. We previouslyexpressed the heterologous drug markers cat, aphA, and kanin D. radiodurans by duplication insertion, producing highlevels of gene amplification (-30 copies per chromosome)without any increase in selective drug concentration (7, 17,18). The heterologous sequence (typically a drug marker inan E. coli plasmid) plus one copy of the flanking hostsequence are repeated in tandem and together are termed theamplification unit (AU) (6) (Fig. 3).Because D. radiodurans is polyploid, it is not known

whether duplication insertions and amplifications are ashared property of chromosomes or limited to a subset. Weexamined this question by use ofjunction fragments, that is,DNA restriction fragments that encompass both a portion ofthe duplication insertion and a portion of flanking DNA.Specific predictions regarding the sizes of the junction frag-ments for any given adjacent insertion may be made. For thispurpose, two neighboring fragments ofD. radiodurans chro-mosomal DNA (one fragment designated mno and the otherdesignated pqrs) were subcloned from pEL2, the mno frag-ment in pPG80 and pPG81 and the pqrs fragment in pPG83(Fig. 4). D. radiodurans wt and rec30 were transformed withpPG80 or pPG81 and selected for Cmr. To obtain chromo-somal DNA from transformants, each was grown individu-ally in TGY broth containing 2 ,ug of chloramphenicol per ml(a strongly selective chloramphenicol concentration for D.radiodurans) to a density of 108 CFU/ml. It was ascertainedthat all bacteria in these cultures were Cmr by platingaliquots on both nonselective and chloramphenicol-selectiveagar, which showed equal numbers of CFU. In addition, 50

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zo 0

0

z

I-6

0 .01 .1 1100o .01.11100o '.01.111DNA (ug/mi)

FIG. 1. Transformation of D. radiodurans strains. The horizontal axis shows the concentration of transforming DNA. The vertical axisshows the frequency of transformation determined by plating on TGY agar containing selective drug concentrations of 25 ,ug of rifampin, 3,ug of chloramphenicol, or 0.003 ,g of MMC per ml, as indicated below. D. radiodurans strains: wt, O; rec30, 0; 30/1, A; 30/wt, A; 112, E.(A) Transformation to Rif' by homologous high-molecular-weight DNA from Rif' isolate R1-R. Without transforming DNA, or in the case of112 recipients, the frequency of Rifr isolates was <10-8. Also plotted is the natural transformation of rec30 to MMC resistance by genomicDNA from wt (V) or rec30 (K). (B) Transformation of D. radiodurans to Cmr by the nonreplicating duplication insertion construction pEL2(Cmr). In the absence of pEL2, or in the case of 112 recipients, the frequency of Cmr isolates was <10-9. In this experiment and all followingexperiments, Cmr rec30 duplication insertion transformants were individually verified as bona fide rec30 by replica plating on MMC-selectiveagar to document the extreme sensitivity to MMC which is characteristic of rec30. (C) Plasmid transformation of D. radiodurans to Cmr bythe autonomously replicating plasmid pI3. In the absence of p13 DNA, or in the case of 112 recipients, the frequency of Cmr isolates was<1o-9.

colonies from each nonselective plate were individuallystreaked on chloramphenicol-selective plates, and all iso-lates were found to retain Cmr.

Bacteria were harvested from the broth, and chromosomalDNA was purified and cleaved with ClaI. Amplification wasreadily observed in the ethidium bromide-stained gel (Fig. 5)as the 5.1- and 2.7-kb bands characteristic of the AU (Fig.4B). This was true for both wt and rec30 transformants, aswell as for those transformed with either pPG80 or pPG81(both of which are identical except for opposite orientationof pELl). Probing of nontransformed Cms isolates withprobe 2 (Fig. 4A and B) showed only the normally present16-kb ClaI chromosomal fragment (Fig. 4B and 5). ProbingofpPG80- and pPG81-transformed Cmr isolates showed in allcases the 9.4-kb junction fragment predicted for homologousduplication insertion of either pPG80 or pPG81 (Fig. 4B and5). Surprisingly, in some wt and rec30 Cmr transformants,

there coexisted the 16-kb ClaI chromosomal fragment typi-cal of nontransformed cells. In no case, however, did anyCmr transformant fail to contain the 9.4-kb junction fragmentindicative of homologous duplication insertion. These re-sults show that most chromosomes contain amplified dupli-cation insertions of pPG80 or pPG81, while some chromo-somes do not contain any duplication insertion.wt and rec30 D. radiodurans also underwent transforma-

tion with pPG83 followed by chloramphenicol selection andgrowth of individual isolates in chloramphenicol-containingTGY broth. As described above, all CFU in broth wereshown to be Cmr. Genomic DNA from transformants waspurified and cleaved with HindIII. Amplification was readilyobserved in the ethidium bromide-stained gel (Fig. 6) as the9.2-kb band typical of the AU (Fig. 4C) in both wt and rec30transformants. Probing of nontransformed Cms isolates withprobe 1 showed only the normally present 23-kb HindIII

-2'Ia -2

0 -3

1 0 20 30-4 II ' ' i

0 200 400 0 500 1000 0 2 4

MMC (ug/ml/min) UV (J/m2) Gy (102)FIG. 2. Survival of strains following exposure to MMC (left), UV (center), and gamma rays (right). The inset in the left-hand panel has

an expanded horizontal axis to demonstrate survival of rec30 and 112, which are quite sensitive to MMC. Gamma radiation doses of greaterthan 500 Gy were not attempted. Symbols for D. radiodurans strains are as for Fig. 1.

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TABLE 2. Uptake of DNA by D. radioduransa

DNA (ng/2.5 x 10' cells)b

Strain Linear Plasmid

Before After Before After

wt 20.2 6.5 7.6 0.6rec30 14.8 8.6 5.1 0.4recll2 210.0 29.9 217.8 11.5

a Cells were prepared for transformation as described in Materials andMethods. After thawing, 50 ,ul of suspension (5 x 107 cells) was added tomicrocentrifuge tubes, to these tubes 3 pI of water or an aqueous solution oflinear chromosomal or circular p13 DNA was added, and the solution wasgently mixed. DNA was metabolically labeled with [5-methyl-3H]thymidine,and the specific activity of both chromosomal and circular p13 DNA was 1.7x 104 cpm/p.g. Either 6 ,ug of chromosomal DNA or 1.5 ,ug of pI3 was added.Tubes were held on ice for 15 min and then transferred to 32°C for 60 min withgentle agitation. Cells were spun down and washed with 300 ,u1 of TGY brothand resuspended in 50 p.1 of TGY with or without 50 p.g of DNase I per ml.These aliquots were incubated at 25°C for 25 min, spun down, and washedwith 300 pI ofTGY. Cells were resuspended in 50 RI of 10 mM Tris (pH 7.5)-imM EDTA, and radioactivity was determined in a scintillation counter.

b Before and after, before and after DNase I treatment.

chromosomal fragment (Fig. 4C and 6). Probing of pPG83-transformed Cmr isolates showed in all cases the 7.2-kbjunction fragment predicted for homologous duplication in-sertion of pPG83 (Fig. 4C and 6). However, in some wtand rec30 pPG83 transformants, there coexisted the 23-kbHindIII chromosomal fragment typical of nontransformedcells. Again, like pPG80 and pPG81 transformants, nopPG83 Cmr transformant failed to contain the 7.2-kb junctionfragment indicative of homologous duplication insertion.These observations are similar to those pertaining to trans-formation by pPG80 and pPG81: following duplication inser-tion of pPG83, most chromosomes contain the amplifiedinsertion, while others do not contain a duplication insertion.

Multimeric circular AUs. We have previously shown thatamplified duplication insertions in D. radiodurans give riseto circular nonreplicating extrachromosomal AUs, identicalin sequence to one linear AU in the chromosome (7, 17).These circules presumably arise from intrachromosomalrecombination between adjacent repeats within amplifiedregions (Fig. 3, steps 4 and 5, left). One explanation for theheterozygosity shown above is that amplified duplicationinsertions are unstable and may be eliminated from thechromosome by intrachromosomal recombination betweendistant repeats within the region of amplification (e.g., Fig.3, steps 4 and 5, right). If so, the likely product of recombi-nation would consist of extrachromosomal circles that con-tain multiple AUs. To determine whether multimeric circularAUs occur, we employed a small AU, so that multimerscould be readily resolved by conventional gel electrophore-sis. Tn9 consists of a central 1,102-base sequence thatcontains cat and two 768-base flanking direct repeats of IS].Tn9, as well as Tn5, has no transposing activity in D.radiodurans (19). There is a single EcoRI site in Tn9, locatedwithin the cat gene (1). pUC19 that contained a Tn9 insertionwas cleaved in the solitary HindlIl site ofpUC19 and ligatedwith a HindIII digest of D. radiodurans genomic DNA, andthe mixture was used to transform D. radiodurans recipientsto Cmr. Amplification of the cat gene occurred via the IS1repeats, rather than the host sequence repeats. Chromo-somal DNA from a Cmr transformant was partially or fullydigested with EcoRI, electrophoresed, blotted, and probedwith pEL1, which contains cat (Fig. 7). In chromosomalDNA fully digested with EcoRI, hybridization was limited to

Homologous pairing

.. - ..

L2J DuplW

Tmnsforming construct

*ab c d * Chromosome

IChromosome

cation Inserton* Amplification unit

abcd cat bcd cat bid c bcde[j] Amplification In chromosome

[J Intrachromosomal Irecombinatdon

IJb cd

_bc cd

b c d c1.

b cdc

Circular camplification b

cunits

FIG. 3. Schematic of duplication insertion and production ofintracellular circular AUs. The heterologous drug resistance deter-minant (cat) on an E. coli plasmid (thick segment) may be integratedinto the D. radiodurans chromosome by in vitro ligation of hostDNA sequences (thin segment bcd) to the E. coli plasmid prior totransformation. After uptake by D. radiodurans, the recipient-derived sequence contained within the chimeric donor plasmidpermits homologous pairing (x) with the host chromosomal se-quence abcde and recombination results in integration of the heter-ologous sequence. This event results in a direct repeat of the hostsequence (bcd with an arrow immediately above) flanking theheterologous drug resistance determinant. The presence of flankingrepeats facilitates gene amplification under selective pressure (6,14), and insertion by this means in D. radiodurans is followed byextensive amplification of the drug resistance determinant plus thechromosomal flanking sequence (AU). Intrachromosomal recombi-nation between repeats could produce monomeric (left) or multi-meric (right) circular forms of the amplification unit. Recombinationmay occur between repeats of the heterologous sequence, or, as

illustrated, between repeats of the host sequence bcd.

a 1.9-kb band, the predicted size for amplification of Tn9 viathe IS] repeats (1.9 kb equals the central sequence plus one

IS) (Fig. 7). The partial EcoRI digest of chromosomal DNAshowed a series of multimers of 1.9, 3.8, and 5.7 kb, and so

on, indicating the presence of amplified linear repeats in thechromosome with a 1.9-kb AU (Fig. 7). Supercoiled DNAwas purified from a transformant by two rounds of isopycnicsedimentation, gel electrophoresed, and probed with pELl.A series of supercoils corresponding to 1.9, 3.8, 5.7, and 7.4kb, and so on, were observed (Fig. 7). The presence ofhighly multimeric extrachromosomal circular AUs is consis-tent with the deletion of large regions of chromosomalamplification in a single step involving intrachromosomalrecombination of nonadjacent repeats (e.g., Fig. 3, steps 4and 5, right).

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BcH5XbE\8 E

pEL24.5 kb 6.0 kb

m n o f p q r a1WBE !:E5 I B CR

8 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IIWEE

Bir E~Jb C' pPG81 H sHProbe3 1

Probe 3 4

"ImIE pPG83

9.4kb7.2 kb

FIG. 4. Detection ofjunction fragments. (A) Duplication insertion constructs pEL2, pPG80, pPG81, and pPG83 and probes for identificationofjunction fragments. A 10.5-kb EcoRI fragment of chromosomal DNA that contains a single internal EcoRI site (top) was obtained from pEL2.The 4.5-kb EcoRI fragment and the adjacent 6.0-kb fragment are identified (mno and pqrs, respectively). The adjacent fragments were separatelycloned in the E. coli vector pEL1, the mno fragment in pPG80 and pPG81 (identical except for opposite orientation of pEL1) and the pqrsfragment in pPG83. Segments: open, pEL1; hatched, pELl BamHI-BclI-A fragment; closed, D. radiodurans sequences; shaded, fragments usedfor hybridization probes. Probe 1, HindIII-SphI fragment from pPG80 or pPG81; probe 2, SmaI fragment from pPG83; probe 3, HindIII-EcoRIfragment from pELl. Restriction endonuclease sites: Bc, BclI; Bc/B, BclI-BamHl fusion; B/E, blunt-end BamHI-EcoRI fusion; C, ClaI; C', ClaIsites that do not cleave in DNA isolated from dam' E. coli because of methylation; E, EcoRI; H, HindlIl; Sp, SphI; Xb, XbaI. (B) Schematicof chromosomal DNA before and after duplication insertion of pPG80 or pPG81 and predicted junction fragments. In a ClaI digest ofnontransformed D. radiodurans chromosomal DNA, probe 2 hybridizes to the pq portion of a normally present 16-kb ClaI chromosomalfragment. A duplication insertion ofpPG80 or pPG81 (which contain the mno sequence) is illustrated below the chromosome as an insert (dashedlines) to the immediate left of the chromosomal mno sequence. Amplification of the duplication insertion is shown, with tandem duplication ofpELl plus mno. Because of the Clal sites in the pELl portion of pPG80 and pPG81, in a ClaI digest of chromosomal DNA of an isolatecontaining the duplication insertion, probe 2 hybridization to the chromosomal pq region identifies a shorter fragment of 9.4 kb. This fragmentis of the same length regardless of whether pPG80 or pPG81 is used for duplication insertion, since the ClaI sites in pELl are symmetrical withrespect to the pELl BamHI site used in cloning fragment mno. (C) Schematic of chromosomal DNA before and after duplication insertion ofpPG83 and predicted junction fragments. In a Hindlll digest of nontransformed D. radiodurans chromosomal DNA, probe 1 hybridizes to theo portion of a normally present 23-kb HindlIl chromosomal fragment. A duplication insertion of pPG83 (which contains the pqrs sequence) isillustrated below the chromosome as an insert (dashed lines) to the immediate right of the chromosomal pqrs sequence. Amplification of theduplication insertion is shown, with tandem duplication of pELl plus pqrs. Because of a Hindlll site in the pELl portion of pPG83, in a HindlIldigest of chromosomal DNA of a strain containing the duplication insertion, probe 1 hybridization to the chromosomal o region identifies a

shorter fragment of 7.2 kb.

A

B

C

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TRANSFORMATION PROPERTIES OF D. RADIODURANS 6115

C0 0 E(X X x X L- EL X MC X X X a. CL L CLOoo0LX0 0 K K K K9 19 9 _ x xXg 9E 8C@ x xX x

X X x x X X M X X ELw X, IL EL O_oo ~o 0o o ~o o o o o .o L CLfi a L

0 0 0 0 0 0 00(0 0 0 0a 0 d)K

-9.2 kb

-5.1 kb

-- 2.7 kb

23.1 -.o ._ ~ _

9.4 -

6.6

4.4-

--16 kb23.1

K'-9.4 kb p

9.4 _go _, m w m -__

5- 5.1 kb6.6 -

-----23 kb

1.

-'-` 0 -9.2 kbmin0M -7.2kb

4.4-

-2.7 kb2.32.0-

FIG. 5. Ethidium bromide-stained agarose gel (top) and autora-diogram of Southern blot (bottom) of ClaI digests of wt and rec30 D.radiodurans DNA containing duplication insertions of pPG80 or

pPG81. All lanes contain 0.5 ,ug of DNA, except the two lanes of wtDNA, which contain 2.0 ,ug. The blot was hybridized against probe2 (per Fig. 4B). There is nonspecific cross-hybridization of probe 2to the 5.1- and 2.7-kb bands, because both are present in very largequantities, because of amplification. Markers (lane M) were restric-tion fragments of HindIII-cleaved phage A DNA. Marker sizes are inkilobases. Chromosomal DNA of nontransformed rec30 producedresults identical to those shown for DNA from nontransformed wtcells, i.e., a single 16-kb band (not shown).

DISCUSSION

Stability of gene amplification. All wt and rec30 Cmrisolates analyzed following transformation with pPG80,pPG81, and pPG83 showed homologous recombination, asdetected on Southern blots by the presence of the appropri-ate junction fragments. However, in many isolates therecoexisted chromosomal fragments typical ofnontransformedcells, although in no case did a Cmr isolate fail to contain thejunction fragment indicative of homologous duplication in-sertion (Fig. 4 through 6). Since D. radiodurans containsmultiple chromosomes, one interpretation is that some chro-mosomes contain duplication insertions while others do notand that it is not necessary for all chromosomes to containthe duplication insertions for expression of Cmr. Since allisolates were substantially amplified for the insertion, asshown by the ethidium bromide stains (Fig. 4 through 6),some chromosomes must contain high levels of amplifica-tion, even though others do not contain any duplicationinsertion at all. This suggests chromosomal instability ofamplified duplication insertions. Evidence in favor of thisproposition was obtained in experiments employing amplifi-cation of Tn9 via IS] direct repeats. It was found thatextrachromosomal AUs can be multimeric, containing as

many as 10 or more copies (Fig. 7). Chromosomal instability

2.32-0FIG. 6. Ethidium bromide-stained agarose gel (top) and autora-

diogram of Southern blot (bottom) of HindIII digests of wt and rec30D. radiodurans DNA containing duplication insertions of pPG83.All lanes contained 0.5 to 1 ,ug of DNA, except the two lanes of wtDNA, which contain 2.0 ,ug. The blot was hybridized against probe1 (per Fig. 4C). In addition, there is faint hybridization to thefull-length 9.2-kb AU because of earlier probing with probe 3 (perFig. 4C). For the markers (lane M), sizes are in kilobases. Chromo-somal DNA of nontransformed rec30 produced results identical tothat shown for DNA from nontransformed wt cells, i.e., a single23-kb band (not shown).

of amplified duplication insertions does not imply instabilityat the cellular level, as we have previously noted that loss ofdrug resistance requires >20 generations in liquid culture inthe absence of selection (17). Extrachromosomal AUs maybe a substrate for recombination with chromosomes, andexchange among chromosomes and extrachromosomal AUsin the presence of drug selection may achieve a dynamicintracellular equilibrium.

Properties of strains rec3O and 112. rec30 shows dimin-ished natural transformation and duplication insertion fre-quencies compared with wt, but plasmid transformation isnormal. Although transformation efficiency is low in rec30,transformants to wt are readily obtained, restoring fullresistance to DNA damage and wt transformation effi-ciencies (Fig. 1 and 2). The rec30 mutant phenotype-sensitivity to DNA damage and defective chromosomaltransformation-suggests that the rec30 gene product acts atthe chromosomal level to promote recombination, whetherfor purposes of transformation or DNA repair.

Strain 112 is totally defective in all forms of transforma-tion, including plasmid transfer, despite apparently in-creased uptake of circular and linear DNA (Fig. 1; Table 2).Strain 112, like rec30, displays sensitivity to MMC and UV(Fig. 2), and we have shown that at higher doses than those

0mlmp4m

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1 2 3 4

ier~~~~~~~~~j....:..:....:era,metrs .Xo-::

5-mer-.. _i4-mer 0*:3-mer-- *

Dimer-"-.. . ..................

-.* Well Higher_ S.nwr Oligomers0- 7-mer~- 6-mer

-0-- 5-mer-.-o 4-mer

_a 3-mer

-*- Dimer

.-a Monomer_

0

0

c

3-

Cf

FIG. 7. Amplification of Tn9 in D. radiodurans Rl. After gel electrophoresis, samples were blotted and probed with pELl. Results areshown for chromosomal DNA partially (lane 1) or fully (lane 2) digested with EcoRI and for chromosomal DNA (lane 3) and supercoiled DNA(lane 4) without restriction enzyme digestion. Markers were Hindlll-cleaved phage A DNA restriction fragments (not shown).

employed here, 112 is more sensitive than wt to gammaradiation and defective in double-strand break rejoining (4).Strain 112 was derived by mutagenesis of strain 302 (Table1), which is defective in mtcA. mtcA encodes an endonucle-ase that recognizes MMC adducts, and 302 is, therefore,sensitive to MMC and certain other adducts (11, 20). How-ever, 302 shows none of the other phenotypic traits of 112:strain 302 has wt resistance to UV and gamma radiation andwt transformation properties (references 11 and 12 andunpublished observations). Unfortunately, reversal of themtcA mutation in 112 is not currently possible, since strain112 is nontransformable and thus far efforts at other meansof genetic exchange, including conjugation, protoplast fu-sion, and transduction, have not been successful (9, 16).Consequently, it cannot be ruled out at this time that thepleomorphic phenotype of 112 somehow requires the pres-ence of the mtcA defect.The phenotypes of rec30 and 112 bear similarities to those

of mutants of other better-characterized naturally transform-able bacteria (8, 15). As pointed out by Morrison andcoworkers (8) with respect to Streptococcus spp., the exist-ence of Rec- mutants that are defective in chromosomaltransformation and resistance to DNA damage (like rec30)and of other Rec- mutants that are, in addition, defective inplasmid transformation (like 112) suggests that recombina-tion is associated with plasmid transfer and is partiallydistinct from recombination functions required for integra-tion of chromosomal markers. Thus, the existence of strain112 suggests that D. radiodurans, like many well-studiednaturally transformable bacteria (2), may specifically de-grade both linear and circular transforming DNA uponuptake and that successful plasmid transfer therefore re-quires recombinational functions.

ACKNOWLEDGMENT

This work was supported by USPHS grant GM39933.

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