Vol. 175, No. 17JOURNAL OF BACTERIOLOGY, Sept. 1993, p. 5411-54190021-9193/93/175411-09$02.00/0Copyright X 1993, American Society for Microbiology

A Rapid Method for Cloning Mutagenic DNA Repair Genes:Isolation of umu-Complementing Genes from Multidrug

Resistance Plasmids R391, R446b, and R471aCHAO HO, OLGA I. KULAEVA, ARTHUR S. LEVINE, AND ROGER WOODGATE*

Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and HumanDevelopment, National Institutes of Health, Bethesda, Maryland 20892

Received 18 February 1993/Accepted 13 June 1993

Genetic and physiological experiments have demonstrated that the products of the umu-like operon are

directly required for mutagenic DNA repair in enterobacteria. To date, five such operons have been cloned andstudied at the molecular level. Given the apparent wide occurrence of these mutagenic DNA repair genes inenterobacteria, it seems likely that related genes will be identified in other bacterial species and perhaps evenin higher organisms. We are interested in identifying such genes. However, standard methods based on eitherDNA or protein cross-hybridization are laborious and, given the overall homology between previouslyidentified members of this family (41 to 83% at the protein level), would probably have limited success. Tofacilitate the rapid identification of more diverse umu-like genes, we have constructed two Escherichia colistrains that allow us to identify umu-like genes after phenotypic complementation assays. With these twostrains, we have cloned novel umu-like genes from three R plasmids, the IncJ plasmid R391 and two InclIMplasmids, R446b and R471a.

Mutagenesis in enterobacteria is dependent upon thefunctional activity of the Umu-like family of mutagenesisproteins (for recent reviews, see references 13 and 58).These mutagenesis proteins are induced as part of thecellular SOS response to DNA damage. Over the past fewyears, molecular and genetic studies have revealed umu-likegenes and proteins in a number of bacterial strains (27, 45).Interestingly, these mutagenic DNA repair genes appear toreside in two locations: either chromosomally, such as theumuDC genes from Eschenichia coli and Salmonella typh-imurium (20, 37, 49, 53), or on certain naturally occurringplasmids, such as the mucAB, impCAB, and samAB operons(23, 30, 37). DNA sequence analysis of these genes hasrevealed that although they vary in their overall DNA andpredicted protein sequences, they are clearly related. In-deed, certain regions are 100% conserved, indicating possi-ble functional domains of these mutagenesis proteins. Ge-netic experiments have suggested that the Umu-like proteinsact to promote translesion DNA synthesis (4). While theactual biochemical mechanism remains to be elucidated,studies in several laboratories have shown that, as part ofthis process, the UmuD-like proteins undergo a RecA-mediated posttranslational cleavage reaction to a mutageni-cally active form (UmuD'-like) (6, 15, 29, 46, 57). Further-more, Echols and colleagues have recently shown thatUmuD', together with UmuC, RecA protein, and DNApolymerase III holoenzyme, is able to promote translesionDNA synthesis of a single abasic lesion in vitro (40).

Recently, growing evidence from a number of laboratorieshas demonstrated that DNA repair pathways, like manyother critical biochemical processes, have been highly con-served throughout evolution (3, 7, 11, 33, 48, 51). In somecases it has been possible to clone eukaryotic and evenmammalian DNA repair genes from the ability of the cloned

* Corresponding author.

DNA to complement certain E. coli phenotypes (8, 34, 41,43). Given the apparent conservation of DNA repair pro-cesses and the wide distribution of the Umu-like proteins ingram-negative bacteria, it seems reasonable to expect thatsimilar mutagenesis proteins might be identified in otherspecies. To facilitate the identification of such genes, wehave developed two E. coli tester strains that allow us toidentify DNAs encoding mutagenic DNA repair proteinsfrom the complementation of certain Umu phenotypes.Using these tester strains, we have cloned the mutagenic

DNA repair genes from three conjugative R plasmids: theIncJ plasmid R391 and two IncL/M plasmids, R446b andR471a. Given our success at cloning umu-complementinggenes from these R plasmids and the fact that DNA repairgenes appear to have been conserved throughout evolution,it may be possible to use these tester strains to isolateumu-like genes from other bacterial species and perhapseven more complex organisms.

MATERIALS AND METHODS

Plasmids. The relevant characteristics of the plasmidsused in this study are described in Table 1. Plasmid pRW178was constructed by subcloning a partial PvuII-NaeI frag-ment from pBluescript KS' with BglII linkers into a deletionderivative of pBR322 which had the region between theunique EcoRI site (at bp 1) and the Eco47III site (at bp 1727)of pBR322 removed and replaced with a unique BglII site.This medium-copy-number plasmid allows LacZ a-comple-mentation and has all the unique restriction sites of thepBluescript KS' plasmid polylinker with the exception ofPstI, which has two sites. R plasmids were kindly providedby R. J. Pinney and transferred into RW96 (see below) bystandard methods of bacterial conjugation (25).

Construction of umu tester strains. The umu tester strainsRW96 and RW126 were constructed by standard methods ofP1 transduction (25). The bacterial strains used in theirconstruction are listed in Table 2.

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TABLE 1. Plasmids used in this study

Plasmid Relevant characteristics Source or reference

R391 Kanr, IncJ R. J. PinneyR446b Tetr, IncLlM R. J. PinneyR471a Ampr, IncLVM R. J. PinneypGB2 Spcr, low-copy pSC101 derivative with mp8 polylinker 9pBluescript KS' Ampr, high-copy plasmid containing the LacZ aL polypeptide and super polylinker StratagenepRW178 Ampr, medium-copy plasmid containing the LacZ a polypeptide and super polylinker This studypRLH429 Spcr, 3.5 kb of R391 DNA cloned into pGB2 This studypRLH430 Ampr, 3.5-kb SmaI-SalI fragment from pRLH429 cloned into Sma- and SalI-digested pRW178 This studypRLH512 SpcT, 4.5 kb of R446b DNA cloned into pGB2 This studypRLH515 Ampr, 4.5-kb EcoRI fragment from pRLH512 cloned into EcoRI-digested pRW178 This studypRLH605 Spcr, 5.8 kb of R471a DNA cloned into pGB2 This studypRLH606 Ampr, 5.8-kb EcoRI-HindIII fragment from pRLH605 cloned into EcoRI- and HindIII- This study

digested pBluescript KS'pRLH608 Spcr, 3.8 kb of R471a DNA. Identical to pRLH605 except that -2 kb of DNA 5' to the umu- This study

complementing genespRW144 Spcr, 2.4-kb mucAB BamHI fragment from pRW72 (15) cloned into pGB2 This studypRW154 Spcr, 2.8-kb umuDC EcoRI fragment from pRW30 (56) cloned into pGB2 This study

Screening for a Umu+ phenotype with a colorimetric papil- whereas the umu-complementing clones gave rise to 20 to 40lation assay. A simple and fast way to screen for a strain papillae (Fig. 1). This dramatic difference between positiveproficient for mutagenesis is to assay for reversion of the and negative clones often allowed us to identify a singlegalK2 allele. We chose to monitor reversion of the galK2 positive clone among a background of several hundredallele with a papillation assay primarily because of a previ- negative ones.ous study that used a similar assay to identify strains that UV survival. Qualitative UV survival data were obtainedhad altered recombinational activity (12). Although both of by streaking an inoculating loop of a fresh overnight cultureour tester strains carry the galK2 allele, RW126 also carries across an LB-agar plate and exposing portions of the streak1ex471::TnS, which results in constitutive expression of all to increasing UV fluences. Although both of the testerLexA-regulated genes. In the recA718 lexA71::TnS back- strains, RW96 and RW126, could in principle be used for thisground, a Umu+ phenotype results in modest spontaneous assay, we found that RW96 gave a much larger differentialmutator activity (52). When plated on MacConkey agar between the Umu+ and Umu- phenotypes. In a typicalindicator plates containing 1% galactose and incubated for experiment, a streak of RW96 exposed to greater than 8 J/m2an appropriate period of time, strains proficient in spontane- gave rise to very few or no viable bacteria, whereas RW96ous mutator activity gave rise to many more Gal' papillae harboring a umu-complementing plasmid was resistant tothan those that were defective for mutagenesis. Best results UV doses of up to 40 J/m2.were obtained when transformation mixes were diluted and For quantitative UV survival curves, bacterial culturesplated directly on MacConkey-galactose plates supple- were grown to a density of 1 x 10i to 2 x 108/ml, harvested,mented with spectinomycin (50 Fg/ml) so that there were 50 resuspended in 10 mM MgSO4, and irradiated with 254-nmto 75 transformants per plate. RW126 grew relatively slowly UV light; appropriate dilutions were plated in triplicate onand gave rise to small, orange-red papillae that took 8 to 10 LB-agar plates. Surviving colonies were scored after over-days of incubation at 37C to develop. The control strain and night incubation at 37°C.negative clones usually gave two to three Gal' papiliae, Quantitative UV mutagenesis assays. The ability of the

TABLE 2. E. coli strains used in this study

Strain Relevant genotype Source° or reference

JM18 recA718 sul4211 thr-I leuB6 A(gpt-prA)62 hisG4 argE3 thi-I galK2 ara-14 xyl-5 Evelyn Witkinmtl-l tsx-33 rpsL31 supE44

RW82 A(umuDC)S9S::cat 56RW96 As JM18 but A(umuDC)595::cat JM18 x P1.RW82GE2084 thr-l leuB6proA2 hisG4 argE3 thi-l lacYl galK2 araD139 xyl-S mtl-l tsx-33 rpsL31 George Weinstock

supE44DE1242 sulA410::TnSpyrD Don EnnisRW117 As GE2084 but suL4lOO::TnSpyrD GE2084 x P1. DE1242DE1776 sulA211 pyrD+ Don EnnisRW118 As RW117 but suU4100::TnSpyrD replaced by suL4211 pyrD+ RW117 x P1. DE1776RW120 As RW118 but also A(umuDC)S95::cat RW118 x P1. RW82DE1918 recA718 srIC300::TnlO Don EnnisRW122 As RW120 but also recA718 srlC300::Tn1O RW120 x P1. DE1918JL1047 k1A71::Tn5 John LittleRW126 As RW122 but also exA71::Tn5 RW122 x P1. JL1047

a Bacterial strains were constructed by standard methods of P1 transduction (25). Recipient strains are indicated first, followed by the source of the P1transducing lysate.

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FIG. 1. Example of the Gal' papillation assay. The figure depictstwo representative bacterial colonies grown on MacConkey indica-tor plates supplemented with 1% galactose and spectinomycin (50p.g/ml) for 10 days at 37°C. The panel on the left is the control testerstrain RW126. The panel on the right is the same tester strainharboring a umu-complementing plasmid. Gal' papillae appear as

dark red spots on the lighter pinkish background.

intact R plasmids and the smaller pGB2-derived recombi-nants to restore mutagenesis functions was compared bytesting for reversion of the hisG4(0c) allele. Bacterial cul-tures were treated as described above for the quantitativeUV survival assay except that cultures were plated on Davisand Mingioli minimal agar plates supplemented with a traceamount of histidine (1 ,ug/ml) as described previously (4).Mutants were scored after 4 days of incubation at 37°C.Induced mutation frequencies were calculated as describedby Sedgwick and Bridges (44).

Isolation of R plasmid DNA. R plasmids, because of theirlarge size and low copy number, have traditionally beendifficult to isolate by conventional methods. However, we

found that R plasmid DNA of sufficient quantity and purity

(0,O0) 11J. N-

CII) itc-T-c

48t23

9.6-

6.6 -*

4.3 -*-

2.3 -*

2.0

FIG. 2. Analysis of isolated R plasmid DNA. R plasmid DNAwas isolated from RW96/R471a, RW96/R446b, and RW96/R391 witha Qiagen Q-20 column and digested with EcoRI. Digested DNA was

analyzed on a 0.5% agarose gel and stained with ethidium bromide.In this exposure, DNA fragments of less than -1.5 kb are barelydetectable and have been omitted. The arrows indicate the relativepositions of reference DNAs (undigested or HindIII-digested ADNA) in kilobases. From the sizes of these DNA fragments, we

estimate that the sizes of intact R471a, R446b, and R391 are 78, 68,and 75 kb, respectively.

could be isolated with a Qiagen Q-20 column (Qiagen Inc.,Chatsworth, Calif.) (Fig. 2). Usually, 50 to 100 ml of a freshLB broth overnight culture was lysed, and the cellular lysateapplied to a single Q-20 column. DNA was recovered fromthe column according to the manufacturer's instructions.This procedure worked well for a number of R plasmids fromdifferent incompatibility groups (unpublished observations),including the IncJ plasmid R391. R391 has been notoriouslydifficult to isolate and was thought to exist integrated into thehost chromosome (32). Although the purity and yield werenot as high as with the other R plasmids, we were able toisolate some episomal R391 DNA (unpublished data), indi-cating that R391 exists as an episomal plasmid at least part ofthe time.

Strategy for the rapid cloning of umu-complementing DNA.To date, five umu-like operons have been cloned and se-quenced. Many of the earlier attempts to clone umu-likegenes were hampered because of an inability to clone thegenes directly into multicopy plasmid vectors (14, 47). Oncethey were identified, it has often been possible to subclonethe umu-like operons onto multicopy plasmids. Under cer-tain conditions, however, overexpression of the Umu-likeproteins from multicopy plasmids can have deleterious ef-fects on cell survival (24). To avoid any such problems in ourinitial screen for umu-complementing genes, we chose theversatile low-copy-number plasmid pGB2 (9) as our primarycloning vector. A prerequisite of our cloning strategy is thatafter digestion of isolated DNA with a particular restrictionenzyme, the umu-complementing genes remain intact. Wechose EcoRI for this purpose, since none of the previouslycloned umu-like genes contains an EcoRI restriction site.

Isolated R plasmid DNA was digested with EcoRI (Fig. 2),and the fragments were ligated into pGB2 that had beenpreviously digested with EcoRI and treated with calf intes-tinal alkaline phosphatase (Bethesda Research Laborato-ries). The ligation mix was used to transform RW126.Transformed cells were selected on the appropriate agarplates supplemented with 50 ,ug of spectinomycin per ml.Positive clones were usually identified first by the colorimet-ric mutagenesis assay and then by the qualitative survivalassay described above. DNA from a positive clone waspartially digested with Sau3AI, and fragments with an aver-age size of 2 to 4 kb were ligated into BamHI-digested pGB2.umu-complementing clones were identified, and the smallestrecombinant clones were partially mapped with 13 or 14restriction enzymes purchased from either New EnglandBiolabs, Inc., or Bethesda Research Laboratories. Theselow-copy-number plasmids were used in our more detailedphenotypic analysis of the novel umu-complementingclones.To more accurately determine the minimal umu-comple-

menting regions in our clones, DNA fragments from thelow-copy plasmids were cloned into either the high-copyplasmid pBluescript KS' or the medium copy plasmidpRW178, and deletion derivatives were generated by theexonuclease III-mung bean nuclease protocol described byStratagene. Deletion derivatives were sized according to theextent to which insert DNA remained intact, and recombi-nants were transformed into RW96. The minimal region ofDNA that encompassed the umu-complementing DNA wasidentified by the qualitative UV survival assay. Clones inwhich the umu-complementing DNA remained intact in-creased the UV resistance of RW96, whereas those withdeletions extending into the umu-complementing regionwere unable to increase UV survival.

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1Oo

z 10-10

a:

LLU10-2z

D 10-3-

10-4

0 3 6 9 12 0 3 6 9 12UV (JM-2)

FIG. 3. UV survival of RW96 and the same strain harboring either R-plasmids R391, R446b, or R471a, or their low-copy-numberderivatives pRLH429 (derived from R391), pRLH512 (derived from R446b), or pRLH605 or pRLH608 (both of which were derived fromR471a). Survival of the same strain carrying a low-copy E. coli umuDC plasmid (pRW154) is included for comparison. Strains: RW96 (0),RW96/R391 (V), RW96/R446b (0), RW96/R471a (A), RW96/pRLH429 (V), RW96/pRLH512 (M), RW96/pRLH605 (A), RW96/pRLH608 (x),and RW96/pRW154 (A).

RESULTS

Construction of umu tester strains. As part of our studieson the mechanisms of inducible mutagenesis in prokaryoticcells, we became interested in identifying mutagenic DNArepair genes from sources other than E. coli or S. typhimu-rium. Standard methods for identifying related DNA se-quences or proteins are often laborious and furthermore arenot always sensitive enough. For example, antibodies raisedto the E. coli UmuC protein do not cross-react with theMucB protein, which is 55% homologous to UmuC (unpub-lished observations). In contrast, the previously identifiedumu-like operons, when cloned into multicopy plasmids, cancomplement the mutagenesis functions of missense E. coliumu mutants. We reasoned, therefore, that the most effec-tive way to identify novel umu-like operons would bethrough a functional complementation assay.

In an otherwise wild-type background, AumuDC andmissense umu mutants exhibit only a slight sensitization tothe lethal effect of UV light (18, 50, 56). However, a numberof years ago, Witkin and coworkers discovered that, incombination with a recA 718 allele, such mutant cells becamemarkedly UV sensitive (55). Furthermore, UV resistancecould be restored by introducing a medium-copy (55) orlow-copy (Fig. 3) plasmid expressing the E. coli UmuDCproteins into these strains. Why a combination of recA 718and umuDC results in the UV-sensitive phenotype is poorlyunderstood, but it is thought to reflect a requirement forthese proteins in a specialized form of replication after DNAdamage (19, 55). Since the E. coli UmuDC proteins couldrescue the UV-sensitive phenotype of these cells, we rea-soned that analogous proteins might have a similar effect.

Studies with a plasmid in which the mucAB genes werecloned into pGB2 confirmed this expectation (unpublished

results). In addition to their moderate UV sensitivity, therecA718 umuDC strains are also rendered phenotypicallynonmutable. As with the other Umu phenotypes, mutabilitycan be restored to these strains by plasmids expressingUmuDC or analogous proteins. In a recA718 strain thatexpresses all LexA-regulated genes constitutively, i.e., onethat carries a exA(Def) mutation, Umu activity manifestsitself as a modest spontaneous mutator activity (52). Usuallythis activity is monitored quantitatively by monitoring rever-sion of an amino acid auxotrophy. However, certain muta-tions in genes required for sugar metabolism can also be usedto give qualitative estimations of mutagenesis. Reversion ofthese genes has the advantage that mutagenesis functionscan easily be monitored on colorimetric indicator plates (12,25).We therefore constructed two tester strains, RW96 and

RW126, which take advantage of these recA718 umu pheno-types and allow us to rapidly identify novel umu-comple-menting genes (Fig. 1 and 3).

Restoration of mutagenesis functions and increase in UVresistance by certain R plasmids. As might be expected, theK-12 recA718 AumuDC excision-proficient strain RW96 wasnot as UV sensitive as previously reported for the B/rexcision-defective strains (Fig. 3) (55). However, introduc-tion of any of the three R plasmids R391, R446b, and R471a,which had previously been identified as expressing Umu-likefunctions, into RW96 resulted in a dramatic increase in UVresistance (Fig. 3) and restored UV-inducible mutagenesis(Fig. 4; see also Fig. 7 and 9). The result with R391 wassomewhat surprising, since R391 had previously been char-acterized as expressing a UV-sensitizing function (35, 54).The UV-resistant phenotype is, however, unique to therecA718 AumuDC background. Backcrosses of R391 into

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500 bp

RSm11

E CCII

RSp

umu -complementingFIG. 6. Partial restriction map of the 4.5-kb EcoRI fragment in

pRLH512. C, ClaI; E, EcoRV; R, EcoRI; Sm, SmaI; Sp, SspI.There are no BamHI, BglII, HindIII, HpaI, YpnI, MluI, NcoI, orSall restriction sites in the 4.5-kb fragment. PstI gave many smallfragments, and these have been omitted. The -2-kb minimal umu-complementing region, as determined by exonuclease III digestion,is indicated below the map.

which allowed us to subclone the DNA as an EcoRI-HindIIIfragment into pBluescript KS'. Further exonuclease III

I I digestion identified a region of -2 kb in the middle of the0 2 4 6 8 fragment that could complement Umu activity (Fig. 5). This

region does not extend past the unique AgeI site andUV (Jm-2) therefore allowed us to construct a deletion derivative of

UV-induced mutagenesis conferred on RW96 by the pRLH605, termed pRLH608, in which -2 kb upstream ofasmid R471a and two low-copy derivatives, pRLH605 and the umu-complementing region was removed.Data for the control strain RW96 are not shown but no The umu-complementing region from R446b was initiallyis+mutants were obtained at any of the UV doses tested. identified as a -9.4-kb EcoRI fragment. Further subcloningepresent the means for at least three independent exper- yielded a plasmid, pRLH512, that contained -4.5 kb ofrains: RW96/R471a (A), RW96/pRLH605 (A), and RW96/ R446b DNA. Restriction analysis of this plasmid allowed us(*). to subclone the umu-complementing genes as a 4.5-kb EcoRI

fragment (Fig. 6). Derivatives of pBluescript KS+ containingthis 4.5-kb fragment proved to be very unstable and rapidly

lifferent strains confirmed the UV-sensitizing phe- lost the ability to phenotypically complement the AumuDCIt would appear that in the recA718 AumuDC mutation. It would appear that the umu-complementingnd, the UV resistance phenotype conferred by the genes from R446b, when expressed from a high-copy plas-u-complementing genes is dominant over the effect mid, are deleterious to cell survival. To circumvent thesecharacterized UV-sensitizing genes. problems, we cloned the 4.5-kb EcoRI fragment into theg of umu-complementing genes from R471a, R446b, medium-copy plasmid pRW178 to generate pRLH515. Using1. Using the papillation assay, we identified a this plasmid, we identified a region of -2 kb necessary forcooRI fragment from R471a that could complement umu complementation (Fig. 6 and 7).tivity. Further subcloning yielded a plasmid, Our initial screen for umu-complementing genes from5, that contained -5.8 kb of R471a DNA. Restric- R391 that had been completely digested with EcoRI did notrme analysis of this clone indicated that it did not yield any positive clones. Since our assay requires that the-ither EcoRI or HindIII restriction sites (Fig. 5),

500 bp

K A B B NE cc

Hd K H

umu -complementing

FIG. 5. Partial restriction map of the 5.8-kb insert in pRLH605.All of the restriction sites for the following enzymes are shown: A,AgeI; B, BglII; C, ClaI; E, EcoRV; H, HindIII; Hp, HpaI; K, Kpnl;M, MluI; N, NcoI; R, EcoRI; S, SmaI. There are no SalI, BamHI,HindIII, SspI, or EcoRI restriction sites in the 5.8-kb fragment. Inaddition, PstI gave many small fragments which were difficult tomap accurately, and these have been omitted. The EcoRI andHindIII sites shown at the ends of the fragment are from the pGB2vector polylinker. The -2-kb minimal umu-complementing region,as determined by exonuclease III digestion, is indicated below themap.

ccZ G

HO

c) cc

z+

I

150

100

50

Or

UV (Jm-2)

FIG. 7. UV-induced mutagenesis conferred on RW96 by theIncL/M plasmid R446b and the low-copy derivative pRLH512. As inFig. 4, data for the control strain RW96 are not shown. Pointsrepresent the means for at least three independent experiments. 5,

RW96/R446b; *, RW96/pRLH512.

250

c:cc0

> 200

cn0

r 150a-

zHD 1000w0

o 50z+

I

FIG. 4.IncL/M pl.pRLH608.induced HThe data riments. St]pRLH608

several dnotype.backgrouR391 umiof the uni

Cloningand R39-35-kb EUmu acpRLH60'tion enzycontain c

R

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500 bp

M

Sm E Hp N NRH Hp S

umu -complementing

FIG. 8. Partial restriction map of the 3.5-kb insert in pRLH429.E, EcoRV; H, HindIII; Hp, HpaI; M, AMuI; N, NcoI; R, EcoRI; S,Sall; Sm, SmaI. There are no BamHI, ClaI, KpnI, PstI, SalI, SmaI,or SspI restriction sites in the 3.5-kb fragment. The SmaI and SalIsites at the ends of the fragment are from the pGB2 vectorpolylinker. The -2-kb minimal umu-complementing region, as de-termined by exonuclease III digestion, is indicated below the map.

umu-complementing region remain intact, we repeated ourscreen with R391 DNA which had been only partiallydigested with EcoRI. R391 encodes a gene for kanamycinresistance. By selecting for kanamycin- and spectinomycin-resistant transformants, we ensured that all of the recombi-nant plasmids carried R391 DNA. RW126 was unsuitable forthis purpose because it carries the lexA71::TnS allele and istherefore already resistant to kanamycin. Instead, recombi-nant plasmids were transformed into RW96 (Kan'), and all ofthe spectinomycin- and kanamycin-resistant clones weretested for their ability to increase UV resistance in RW96. Anumber of these clones proved positive, and the smallest ofthese was used for further subcloning. After digestion withSau3AI, a recombinant plasmid that contained a -3.5-kbR391 DNA fragment, termed pRLH429, was chosen forfurther analysis (Fig. 8 and 9). Partial restriction mapping ofpRLH429 allowed us to subclone the DNA into the medium-copy plasmid pRW178 as a SmaI-Sail fragment. Deletionanalysis of this plasmid (pRLH430) identified the umu-

complementing region of -2 kb located close to the SaiI endof the fragment (Fig. 8).Phenotpes of the novel umu-complementing plasmids.

Comparison of the three novel low-copy umu-complement-ing plasmids revealed that all three confer UV resistance(Fig. 3), spontaneous mutability (Table 3), and UV-inducedmutability (Fig. 4, 7, and 9) to the recA718 AumuDC strain

ccw

0.

CO

z

+CCI

150

100

50

UV (Jm-2)

FIG. 9. UV-induced mutagenesis conferred on RW96 by the IncJplasmid R391 and the low-copy derivative pRLH429. Values repre-sent the means for at least three independent experiments. V,RW96/R391; V, RW96/pRLH429.

TABLE 3. Spontaneous mutagenesis conferred by R plasmidsand their low-copy-number umu-complementing derivatives

on the umu tester strains RW96 and RW1260

Strain Plasmid His+ mutantsStrain Plasmid ~~~~~(no./plate)RW96 (1ex4A recA718 AumuDC) R471a 6.4

pRLH605 7.4pRLH608 35.9

R446b 11.0pRLHS12 28.6

R391 12.0pRLH429 54.9

RW126 [kxA(Def) recA718 AumuDC] pRLH605 247pRLH608 165

pRLH512 536

pRLH429 2,400

pRW154 98

pRW144 315

a The values represent averages for at least three separate experiments,each with three plates per experiment. Both of the control strains, RW96 andRW126, usually gave, on average, zero or one spontaneous His+ mutant perplate. The level of spontaneous mutagenesis observed when RW126 harborspRW154 (E. coli umuDC) or pRW144 (mucAB) is included for comparison.

RW96 to a greater extent than their respective parental Rplasmids. Since this increase was often between three- andfivefold, it is possible that it could be due in part to anincrease in the copy number of the plasmid from which theumu-complementing proteins were expressed. Alternatively,these phenotypes could also occur from the deletion ofDNAencoding toxic proteins, such as colicins, or potential regu-latory proteins. For example, pRLH605 and pRLH608should be expressed at similar levels, yet pRLH608 is moreproficient at complementing Umu functions than pRLH605.The only difference between these plasmids is that pRLH605still contains the -2-kb region upstream of the umu-comple-menting region.

In contrast to the enhanced spontaneous mutability thatpRLH608 conferred on the lexA' strain RW96, the level ofspontaneous mutagenesis that pRLH608 promoted in thelexA(Def) derivative RW126 was lower than that promotedby pRLH605, with 165 and 247 histidine prototrophs perplate, respectively (Table 3). In comparison, pRLH512 gaverise to 536 histidine prototrophs and pRLH429 gave approx-imately 2,400 prototrophs per plate. All of the plasmidspromoted spontaneous mutagenesis better than the E. coliumuDC genes (pRW154), while pRLH512 and pRLH429appeared to be more efficient than mucAB (pRW144). Sinceall of these umu analogs were cloned in the same vector,these phenotypes are presumably directly related to thephysical properties of the umu-complementing proteins en-coded by these plasmids.

DISCUSSION

We have constructed two new tester strains that haveallowed us to identify rapidly the minimal umu-complement-ing DNA from three large (-68 to 78 kb) multidrug resis-tance R plasmids. These R plasmids were chosen for a

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variety of reasons. First, research by others had suggestedthat they might contain umu analogs (5, 39, 54); second,unlike the IncN plasmid R46, which encodes mucAB; theIncI plasmid TP110, which encodes imppCAB, and the crypticpSLT plasmid encoding samAB (all of which were isolatedfrom Salmonella strains [23, 30, 38]), R391, R446b, andR471a were originally isolated from Providencia rettgeri(10, 36a), Morganella morganii (16, 36a), and Serratiamarcescens (17), respectively, and therefore might haveevolved differently from the previously identified Salmonellaplasmids; and third, they appeared to promote mutagenesisfunctions to various degrees. In general, previous reportshave suggested that R446b is more efficient at promotingmutagenesis functions than R471a (yet both come from thesame plasmid incompatibility group) and both are muchbetter than R391 (5, 28, 54). We hypothesized that if we weresuccessful in cloning the umu-like genes from these plas-mids, our results might provide us with a better insight intothe structure-function relationship between these and thepreviously identified mutagenesis proteins.The preliminary characterization of the novel umu-com-

plementing genes presented here suggests that all appear tohave interesting phenotypes that warrant further study. Forexample, the -2-kb region upstream of the umu-comple-menting genes in pRLH605 might encode a protein thatattenuates their function. The idea that the functional activ-ity of plasmid-encoded Umu analogs is more tightly regu-lated than that of their chromosomal counterparts is notnew. For example, a region that maps -2 kb from themucAB genes that affects mutagenesis functions has previ-ously been identified (22). In addition, the imp operoncontains an extra gene, impC, that appears to encode aregulatory protein (23), while recent studies suggest that theSamAB proteins are not active when expressed from theirnative plasmid pSLT (21) or from a low-copy plasmid (31).This type of attenuation may therefore be common toplasmid-encoded umu analogs.A particularly striking observation in this study is our

finding that R391 did not sensitize either of the tester strainsto the lethal effects of UV light. While the reason for the UVsensitization in a wild-type background is unknown, it doesappear to occur via a recA-dependent pathway (35). Presum-ably the UV-sensitizing proteins cannot function in therecA718 background, allowing the UV resistance phenotypeof the umu-complementing genes to dominate. We consid-ered the possibility that these functions are somehow re-lated, especially as our data and those from a previous studyhave shown that the umu-complementing region and theUV-sensitizing region map close to the kanamycin resistancegene of R391 (36). However, none of our low-copy, umu-complementing plasmids causes UV sensitization in a wild-type background.Another quite unexpected observation was the dramatic

mutator effect that pRLH429 had on the spontaneous muta-bility of the kxA(Def) strain RW126. The level of spontane-ous mutagenesis was much greater than that seen with theother low-copy umu-complementing plasmids and evenhigher than that promoted by the MucAB proteins expressedfrom pGB2. It would appear that when fully derepressed, theumu-complementing genes derived from R391 are the mostefficient of the currently identified umu analogs at promotingspontaneous mutagenesis.We have used the two tester strains to clone umu-comple-

menting genes from three large conjugative plasmids. Thestrains also identified the chromosomal E. coli umuDC geneswhen cloned into the same low-copy vector. Given the

apparent evolutionary conservation of DNA repair genes, itmay be possible to use these strains to identify mutagenicDNA repair operons from other bacterial plasmids andstrains or perhaps even higher organisms. The search forumu-complementing genes is, however, complicated by thefact that not only must recombinant proteins be expressed inE. coli, they must be able to interact with proteins known tobe required for the mutagenic process, such as RecA (1, 12,52). Since the structure and function of RecA-like proteinsseem remarkably conserved throughout a number of pro-karyotic (26, 42) and eukaryotic (2, 33, 51, 59) species, it isreasonable to think that mutagenesis proteins active in aheterologous background might also be active in E. coli.

ACKNOWLEDGMENTSWe thank Evelyn Witkin, Don Ennis, John Little, and George

Weinstock for bacterial strains; Dick Pinney for providing the Rplasmid-containing strains; Adriana Bailone and Raymond Devoretfor suggesting the papillation assay; Ekaterina Chumakov and JanetHauser for helpful discussions; and Fuyen Yip for technical assis-tance in the early stages of this project.

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