Vol. 172, No. 11JOURNAL OF BACTERIOLOGY, Nov. 1990, P. 6223-62310021-9193/90/116223-09$02.00/0Copyright © 1990, American Society for Microbiology

LexA-Independent Expression of a Mutant mucAB OperonKAREN PERRY McNALLY,tt NANCY E. FREITAG, AND GRAHAM C. WALKER*

Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 29 May 1990/Accepted 18 August 1990

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene

products of which are required for the SOS-dependent processing of damaged DNA necessary for mostmutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit,with LexA acting as a direct repressor. pGW16 is a pKMl01 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increasein spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able toenhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includesthe mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence ofa 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in'thatregion, a G. C-to-A T transition that alters a sequence homologous to known LexA-binding sites. DNA gelshift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation,whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also altersan overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closerto the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicellsis increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmidsuggests that this mutation also increases the activity of the mucAB promoter.

Most mutagenesis of Escherichia coli by UV irradiationand a variety of chemicals requires the products of thechromosomally encoded umuDC operon (5, 28, 35, 42, 43,47). Both umuD and umuC mutants are virtually nonmutablewith UV and many chemicals (5, 9, 36). The mucAB operonis a plasmid-borne analog of the umuDC operon (26, 27, 44)and is encoded by the 35.4-kb N-incompatibility groupplasmid pKM101 (33, 46). pKM101 is one of a number ofextrachromosomal elements that enhance both the resis-tance of their hosts to DNA-damaging agents and theirmutagenesis by these agents (37, 42). Because of its mu-tagenesis-enhancing properties, pKM101 has been intro-duced into the Ames Salmonella strains for detecting car-cinogens as mutagens (17).As in the case of umuDC, the repair and mutagenesis

phenotypes conferred by mucAB are dependent on a hostrecA+ lexA+ genotype (40). Like chromosomally encodedSOS loci in E. coli (13, 28, 42, 43), expression of mucAB isinduced by conditions that damage DNA or interfere with itsreplication (6). Genetic analyses of the regulation of mucABexpression are consistent with the interpretation that LexAis the direct repressor of the mucAB operon (6), and we haveidentified a site in the 5'-flanking region of muc that ishomologous to the LexA recognition sites of other SOS loci(26).

In addition to the transcriptional regulation of mucAB andumuDC by the SOS system, both the UmuD and MucAproteins undergo a form of posttranslation modification inSOS-induced cells: a RecA-mediated proteolysis thatcleaves the UmuD protein at its Cys-24-Gly-25 bond and the

* Corresponding author.t Present address: School of Pharmacy, University of California,

San Francisco, Laurel Heights Campus, San Francisco, CA 94143-1204.

t Previous papers by this author were published under the nameKaren L. Perry.

MucA protein at its Ala-25-Gly-26 bond (3, 16, 23, 26, 34; H.Shinagawa, personal communication). In the case of UmuD,this cleavage activates the protein for its role in mutagenesis,the resulting carboxyl-terminal fragment being both neces-sary and sufficient (23). In the case of MucA, RecA-mediatedcleavage has been shown to occur in SOS-induced cells, butit is not yet clear that this cleavage is necessary for MucA tofunction in mutagenesis (16; Shinagawa, personal communi-cation). An ability of the uncleaved MucA protein to carryout its role in mutagenesis with reasonable efficiency mayhelp to explain the observation that recA430 mutants, whichare deficient in the cleavage of both UmuD (3, 23, 34) andMucA (Shinagawa, personal communication), are deficientin umuDC-dependent mutagenesis but proficient in mucAB-dependent mutagenesis (1, 16; Shinagawa, personal commu-nication).TnS and Tn1000 insertion mutants of muc have been

obtained in screens for plasmid derivatives that are unable toenhance base substitution mutagenesis (27, 33). Each ofthese mutants is also unable to enhance resistance to UVkilling. The various muc mutations fall into two groups onthe basis of complementation analyses. Insertion mutationsin mucB prevent the synthesis of the 46,000-molecular-weight MucB protein. Insertion mutations in the promoter-proximal mucA gene prevent the synthesis of the 16,000-molecular-weight MucA protein and are also polar on mucB(27). Both the MucA and MucB gene products have beenshown to be required for expression of the repair andmutagenesis phenotype (27).A pKM101 mutant with a different phenotype was isolated

in a screen for plasmid mutants that confer an increased rateof spontaneous reversion (41). This plasmid, pGW16, de-rived from pKM101 by N-methyl-N'-nitro-N-nitrosoguani-dine mutagenesis, caused a modest increase in the frequencyof spontaneous reversion to His' of a Salmonella typhimu-rium hisG46 strain relative to that caused by pKM101. Inaddition, it increased the susceptibility of the cells to muta-

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genesis by 4-nitroquinoline-1 oxide (41). In this study, we

have investigated the molecular basis of the unusual pheno-type of pGW16 and present evidence that it is due to an

overproduction of the MucA and MucB proteins as the resultof a single-base-pair-substitution mutation that both de-creases LexA binding and increases transcription.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli K-12 strains werethe following derivatives of AB1157 [thr-J leuB6 proA2hisG4 thi-J argE3 lacYJ galK2 ara-14 xyl-5 mtl-1 tsx-33rpsL31 Xrac kdgK51 A(gpt-proA)62 mgl-51 rfbDJ supE44]:TK610 (as AB1157 but arg+ ilv-325 uvrA6 umuC36) (9);DM49 [as AB1157 but lexA3(Ind-)] (22); JC2926 (as AB1157but recA13) (A. J. Clark); and JC7623 (as AB1157 but recB21recC22 sbcB15) (A. J. Clark). The following plasmids havebeen described previously: pACYC184 (4), pKM101 (12, 21,46), pGW16 (41), pGW271 (11), and pSE152 (5). Bacterio-phage M13mp8 (19) was obtained from New England Bio-Labs.

Plasmid pGW1704 contains the mucAB and 5'-flankingregions of pKM101 cloned into the HinclI gap ofpACYC184(see Fig. 4). Plasmid pGW271 (11) was the source of the3.9-kb fragment from the HpaI site of the insertion flS: :TnSto the HpaI-7 site of pKM101. HpaI-restricted pGW271DNA and Hincdl-restricted pACYC184 DNA were ligatedand used to transform TK610 to Cmr. Transformant colonieswere screened for tetracycline sensitivity and UV-inducedmutagenesis to His'. Plasmid DNAs from UV-mutablederivatives were characterized by restriction analyses. Thederivative pGW1704 contained the 3.9-kb HpaI fragmentcloned into the HinclI-gap of pACYC184 in the orientationshown in Fig. 4. TnS insertion derivatives of pGW1704 wereobtained by the procedure of Winans et al. (45). Random TnSinsertion derivatives of pGW1704 were characterized byrestriction analyses, and pGW1704 fQ16::Tn5 was chosen foruse in this study (see Fig. 4). The insertion fQ16::TnS mapsapproximately 0.6 kb upstream of the start of mucA.The insertion fQ16::TnS was recombined into plasmids

pKM101 and pGW16 so as to create convenient restrictionsites in the region upstream of mucAB. The recB recC sbcBstrains JC7623(pKM101) and JC7623(pGW16) were trans-formed to Kmr with EcoRI-linearized pGW1704 fQ16::TnS(45). Plasmid DNAs from the Kmr JC7623(pKM101) trans-formants were purified and characterized by restrictionanalyses. One of the pKM101 derivatives, which appeared tohave acquired the insertion fQ16: :TnS by simple doublehomologous recombination, was retained for further manip-ulations. The plasmid genotypes of Kmr JC7623(pGW16)transformants were determined as follows. Plasmid DNAswere purified and used to transform TK610 and DM49 to AprKmr. TK610 transformants were screened for their resis-tance to UV killing by using qualitative plate assays. DM49transformants were screened for methyl methanesulfonate(MMS)-induced mutagenesis to His' by using qualitativedisk assays. Twenty Kmr pGW16 derivatives were charac-terized in this manner. Five of these derivatives retained theMuc phenotypes of pGW16. One of these five plasmids wasfurther characterized by restriction analyses and appeared tohave acquired the insertion fQ16: :TnS by simple doublehomologous recombination. This pGW16 fQ16::TnS deriva-tive was retained for further manipulations.A 10.9-kb deletion derivative of pKM101 fQ16::TnS was

constructed by ligating the pKM101 Sall-1 site to the Sailsite of the TnS insertion. This derivative was designated

pGW1717. A similar deletion derivative ofpGW16 fQ16: :Tn5was constructed and designated pGW1718. pGW1718 wasshown to retain the Muc phenotypes of pGW16 by qualita-tive UV killing and MMS-induced mutagenesis assays.

Media. LB medium, M9 minimal medium, and F top agarhave been described by Miller (20); nutrient medium hasbeen described by Walker (40). Soft agar is 0.8% nutrientbroth, 0.5% NaCl, and 0.65% agar. When included, specti-nomycin was used at 100 Rg/ml, and kanamycin sulfate,ampicillin, and chloramphenicol were used at 25 ,uglml.Spectinomycin was a gift of the Upjohn Co., adenine, aminoacids, and all other antibiotics were purchased from SigmaChemical Co.Enzymes and standard procedures. DNA polymerase I

(Klenow fragment) was obtained from International Biotech-nologies Inc., and [32P]ATP was obtained from AmershamCorp. All restriction enzymes and DNA ligase were pur-chased from New England BioLabs and were used under theconditions suggested by the supplier. Procedures for thepreparation of plasmid DNAs (29, 38), agarose gel electro-phoresis (14), and transformation of E. coli with plasmidDNA (14) have been previously published.MMS mutagenesis assay. A 0.1-ml sample of a fresh

stationary-phase nutrient broth culture and the appropriateamount of a solution of MMS in dimethyl sulfoxide wereadded to F top agar supplemented with 0.05 mM histidine,100 ,ug of adenine per ml, 2 ,ug of thiamine per ml, and 1 mgof all other required amino acids per ml. Adenine was addedto suppress the partial purine auxotrophy that is conferredby pKM101 (39). The top agar was then poured on minimal-glucose plates, and the number of His+ revertants wasscored after 2 days incubation at 37°C. MMS mutagenesiswas measured qualitatively as follows: cells were plated insupplemented F top agar as described above, and a paperdisk containing 10 ,ul of a 1:10 dilution of MMS in dimethylsulfoxide was placed in the center of the plate.UV killing. Cells grown in LB (plus drug[s] for plasmid-

containing strains) to 2 x 108/ml were centrifuged andsuspended in an equal volume of 0.85% NaCl. Cells (6 ml)were placed in a glass petri dish (100 mm) and irradiatedwith a 15-W General Electric germicidal lamp. After irradi-ation, the cells were diluted in 0.85% NaCl and plated on LBplates in 2.5 ml of soft agar. Plates were incubated at 37°Covernight.

Maxicells and electrophoresis of proteins. Plasmid proteinswere labeled by the maxicell procedure essentially as de-scribed by Sancar et al. (31), with the following modification:the strains bearing plasmids were JC2926 derivatives, andthese strains were irradiated with 35 J/m2. Samples wereelectrophoresed in 14% sodium dodecyl sulfate-polyacryla-mide gels, using the Laemmli buffer system (10).M13 cloning and DNA sequence determination. The se-

quence of the 5' and upstream regions of pGW16-derivedmucAB was determined by using the dideoxynucleotidetermination method (32) as described by Messing et al. (18).pGW1718 DNAs restricted with RsaI were ligated withSmaI-restricted M13mp8 vectors. Recombinant phage werescreened for hybridization to an M13mp8 derivative thatcontains an AluI fragment from the 5' and upstream regionsof pKM101-derived mucAB (26). The recombinant phageused for the nucleotide sequence determination contains a119-bp insert of pGW1718 DNA in M13mp8.DNA fragment gel shift assays. The ability of LexA protein

to bind the potential LexA recognition sequences of themucAB operon of pKM101 and of pGW16 was investigatedby using a gel shift assay (7). Plasmids pGW1717 and

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,ul Methyl Methonesulfonate UV Dose (J/m2)FIG. 1. (A) MMS-induced reversion to His+ of uvrA umuC strains containing plasmids pGW16 and pKM101. Cells were plated on minimal

media in the presence of limiting concentrations of histidine and various doses of MMS. Spontaneous His' revertants were subtracted fromthe total number of revertants on each plate to obtain the number of MMS-induced revertants. There were no MMS-induced revertants ofTK610 (uvrA umuC) without a plasmid. Symbols: A, TK610(pGW16); 0, TK610(pKM101). (B) Survival after UV irradiation of uvrA umuCstrains containing plasmids pGW16 and pKM101. Cells were irradiated at fluences of 0.25 J/m2Is for various lengths of time and plated on LBplates. Symbols: 0, TK610 (uvrA umuC); A, TK610(pGW16); 0, TK610(pKM101).

pGW1718 were first digested with EcoRI restriction enzyme,and the linearized plasmid DNA was then used to generate124-bp fragments from each of the plasmids, using thepolymerase chain reaction (30) with the following two 27-nucleotide primers: CATATAGTAGAGAACCTGTAAAand GTGTACGCTGGCGCCGGAGCTT. The fragmentswere purified after electrophoresis through low-melting-point agarose gels and radioactively labeled at their 5' endsby using T4 polynucleotide kinase as described by Maniatiset al. (14). LexA protein (a gift from J. W. Little) wasincubated for 20 min at room temperature in reaction mixes(20 Ru) containing 7.7 nM (ca. 500 cpm) pGW1717- orpGW18-derived fragments and 150 nM poly(dI-dC) in 5 mMTris hydrochloride (pH 7.5)-5 mM MgCl2-50 ,ug of bovineserum albumen per ml. After incubation, the samples weresubjected to electrophoresis through 5% polyacrylamide gelsin 0.045 M Tris borate-0.001 M EDTA buffer.

RESULTS

Effects of pGW16 on mutagenesis and resistance to UVkilling in an E. coli host. Before further studies of plasmidpGW16 were carried out, the plasmid was introduced into anE. coli uvrA umuC36 strain; this host was chosen becausepKM101-mediated mutagenesis by MMS and pKM101-me-diated resistance to UV killing are more pronounced in thisbackground than in a uvrA+ umuC+ background (44).pGW16 enhanced MMS-induced hisG4 --His+ reversion inan E. coli uvrA umuC host to a greater extent than did

pKM101 (Fig. 1A). The MMS doses used in this assay were

sufficiently low that there was no effect on survival of thestrains.We also examined the ability of pGW16 to increase

resistance to killing by UV in a uvrA umuC background.Despite the fact that the presence of pKM101 in the uvrAumuC strain greatly increases its resistance to killing by UV(44), the presence of pGW16 did not cause an increase inresistance to killing by UV (Fig. 1B). This latter pGW16phenotype does not appear to be due to a mutation thatcauses the plasmid to sensitize its host to UV killing inde-pendently of mucAB activity, since the UV sensitivity ofuvrA umuC strains carrying either pGW16 mucA::TnS orpGW16 mucB: :TnS derivatives is similar to that of theplasmid-free host (data not shown). If pGW16 carried a

mutation outside of the mucAB region which causes host UVsensitization independently of mucAB, then strains carryingthese muc::TnS derivatives of pGW16 would have beenexpected to have been more UV sensitive than were plas-mid-free strains.The phenotype of enhancing MMS mutagenesis but not

increasing resistance to UV killing makes pGW16 differentfrom the other pKM101 mutants that have been isolated todate (27, 33, 41), all of which have lost the abilities both toenhance MMS mutagenesis and to increase resistance to UVkilling. However, a possible clue to the molecular basis ofthis unusual phenotype was provided by our observationthat these effects caused by the presence of pGW16 in an E.coli cell were reminiscent of those caused by the high-copy-

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UV Dose (J/m2)FIG. 2. Survival after UV irradiation of uvrA umuC strains

containing pGW1700 and pKM101. Symbols: 0, TK610 (uvrAumuC); U, TK610(pKM101); A, TK610(pGW1700); O, TK610(pKM101)(pGW1700).

number mucA+B+ recombinant plasmid pGW1700 (27).pGW1700 enhances the MMS-induced mutagenesis of auvrA umuC host to a greater extent than does pKM101 (27).However, it was not as proficient as pKM101 at conferringresistance to UV killing (Fig. 2). Furthermore, the presence

of both pKM101 and pGW1700 in a uvrA umuC strain furtherdecreased the cellular resistance to UV killing almost to thelevel of a plasmid-free strain (Fig. 2). This phenomenonappears to be due to the elevated mucAB gene dosage, sincethe UV resistance of a uvrA umuC strain carrying pKM101and a muc::TnlO00 derivative of pGW1700 is the same as

that of a strain carrying pKM101 alone (data not shown).pGW16 increases MMS mutagenesis and resistance to UV

killing in a lexA3(Ind-) host. The similarity between thephenotypes conferred by pGW16 and those conferred bypGW1700 suggested to us that pGW16 carries a mutationwhich results in increased mucAB expression. Since expres-

sion of the mucAB operon is under the control of the recA+lexA+-dependent SOS regulatory system (6), we first con-

sidered the possibility that the pGW16-encoded mucABoperon is insensitive to repression by LexA. To examine thispossibility, we introduced pGW16 into the lexA3(Ind-)strain DM49. The lexA3(Ind-) allele prevents induction ofthe wild-type operon by agents that induce SOS (42). Theability of the mucA+B+ plasmid pGW1704 to enhance MMSmutagenesis in this background was negligible (Fig. 3A). Incontrast, we found that pGW16 substantially increasedMMS-induced hisG4 --His+ reversion in a lexA3(Ind-) host(Fig. 3A). Furthermore, the mutagenesis-enhancing pheno-

type conferred by pGW16 in a lexA3(Ind-) host was domi-nant to the phenotype conferred by pGW1704 (Fig. 3A).These results are consistent with the interpretation thatpGW16 carries a mutation which weakens LexA binding tothe mucAB operator. Alternative explanations are thatpGW16 carries a mutation which either increases the activityof the mucAB promoter or increases the stability of theMucA or MucB proteins.We also observed that pGW16 increased the resistance of

the lexA3(Ind-) strain to UV killing to a greater extent thandid pKM101 (Fig. 3B). The extent of protection against UVlethality mediated by pKM101 in this background (Fig. 3B)was modest compared with its effect in a lexA+ strain (Fig.1B). This ability of pGW16 to enhance the survival of alexA3(Ind-) host was in marked contrast to the inability ofthe plasmid to enhance the survival of a lexA+ host (Fig.1B). This observation supports the hypothesis that theinherent activity of the mucAB gene products is not alteredby the pGW16 mutation(s) and suggests, rather, that thefailure of pGW16 to make a lexA+ strain more resistant toUV killing is due to an overproduction of the mucAB geneproducts. This interpretation suggests that synthesis of thepGW16-encoded mucAB proteins under the conditions of theassay is lower in the lexA3(Ind-) background than in thelexA+ background and therefore that expression of theoperon in a lexA3(Ind-) host is not fully independent ofLexA control.

Localization of the mutation(s) responsible for the effects ofpGW16 on mutagenesis and resistance to UV killing. Themutation(s) responsible for the effects of pGW16 on MMSmutagenesis and resistance to UV killing was localized by invitro recombination to a 3.75-kb fragment extending from 2.2kb upstream of the mucAB operon to 0.2 kb downstream ofthe mucAB operon. To carry out this genetic mapping, wemade use of a derivative of the mucA+B+ recombinantplasmid, pGW1704, that carried a TnS insertion (designatedfl16::TnS) located 0.6 kb upstream of the start of the mucAgene (Fig. 4). As described in Materials and Methods,pGW1704 carries the region of pKM101 DNA extendingfrom the site of the TnS insertion f1l5::Tn5 (11) (located 2.2kb upstream of the mucAB operon) to the HpaI-7 site (11)(located 0.2 kb downstream of the mucAB operon) (Fig. 4).The insertion Qfl6: :TnS was recombined from pGW1704 intopGW16 in a recB recC sbcB host by the method of Winans etal. (45). Twenty Kmr recombinants ofpGW16 were screenedfor their ability to enhance mutagenesis in a lexA3(Ind-) hostand for their effects on resistance to UV killing in a uvrAumuC host. Fifteen of these recombinants conferred thephenotypes of wild-type pKM101, whereas five of the re-combinants conferred the phenotypes of pGW16. One of therecombinants that conferred a pGW16 phenotype was char-acterized by restriction analyses and appeared to haveacquired the fl16::TnS insertion by a simple double homol-ogous recombination event. Thus, the mutation(s) responsi-ble for the effects of pGW16 on MMS mutagenesis andresistance to UV killing is located within this 3.75-kb frag-ment and is approximately 75% linked to the Qfl6::TnSinsertion under the recombination conditions used in thisexperiment. One of the pGW16 Q1l6::TnS derivatives wasretained, as was a corresponding pKM101 derivative ob-tained by an analogous recombination between pGW1704Q116::TnS and pKM101.The mutation(s) carried by pGW16 was further localized

by deletion analysis to the region on the mucA+B+ side ofthe Q116::TnS insertion. Deletion derivatives of plasmidspKM101 Q116::TnS and pGW16 Q116::TnS described above

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pi Methyl Methanesulfonote UV Dose (J/m2)FIG. 3. (A) MMS-induced reversion to His' of lexA(Ind-) strains containing pGW16 and pGW1704 and showing the dominance of the

pGW16 phenotype. Cells were plated on minimal media in the presence of limiting concentrations of histidine and various doses of MMS.Spontaneous His' revertants were subtracted from the total number of revertants on each plate to obtain the number of MMS-inducedrevertants. There were no MMS-induced revertants of DM49 [lexA(ind-)]. Symbols: A, DM49(pGW16); V, DM49(pGW1704); V,DM49(pGW16)(pGW1704). (B) Survival after UV irradiation of lexA(Ind-) strains containing pGW16 and pKM101. Cells were irradiated ata fluence of 0.25 J/m2/s for various lengths of time and plated on LB plates. Symbols: E, DM49 [lexA(Ind-)]; A, DM49(pGW16); 0,DM49(pKM101).

were constructed by ligating the SalI-1 site of each plasmid(11) to the SaI site of their respective TnS insertions. Thesederivatives were designated pGW1717 and pGW1718, re-spectively (Fig. 4). Each deletion derivative retains thecharacteristics of its parental plasmid with respect to MMSmutagenesis and UV resistance, indicating that the muta-tion(s) responsible for the mucAB phenotype must be lo-cated within the DNA fragment extending from 0.6 kbupstream of the mucAB operon to 0.2 kb downstream of themucAB operon.DNA sequence of the 5' noncoding region of the mutant

mucAB operon. The foregoing observations suggested thatpGW16 carries at least one mutation which results in in-creased expression of the mucAB operon. The effects ofpGW16 on MMS mutagenesis and UV resistance in alexA3(Ind-) strain suggested that a mutation in pGW16might reduce the affinity of the mucAB operator for LexA.We have previously determined the nucleotide sequence ofmucAB+ and have identified potential operator and pro-moter sequences in the 5' noncoding region of the operon(26). To examine whether these sequences are altered in thepGW16 mutant, we cloned a 119-bp fragment containing the5' noncoding region of the pGW16 mucAB operon into thebacteriophage vector M13mp8 and determined the nucleo-tide sequence of the fragment. Comparison of the wild-typeand mutant sequences revealed a single-base difference inthe region between the -96 nucleotide of the 5' noncodingregion and the +23 nucleotide of the mucA gene. Themutation is a G * C --A T transition that alters the highly

conserved third position of the potential LexA recognitionsequence and would be expected to reduce LexA binding(Fig. 5). Overlapping the putative LexA recognition se-quence is a potential -10 promoter region for mucAB. Themutation carried by pGW16 is at the third position of thisregion (Fig. 5). The mutant sequence ofpGW16 more closelyresembles the consensus sequence for E. coli promoters (8)than does the pKM101 sequence, suggesting that the activityof the pGW16-encoded muc promoter may be greater thanthe activity of the pKM101-encoded promoter. Despitenumerous attempts to recombine this mutation into amucA+B+ derivative, we have not been able to detectrecombination between the 119-bp cloned pGW16 fragmentand a mucA+B+ pKM101 derivative under conditions inwhich recombination frequencies of 10-5 or greater wouldhave been detected. The reason for this lack of recombina-tion is as yet undetermined but may be related to theobservations of several investigators that the presence ofpKM101 or its parent, R46, in a cell results in a decreasedfrequency of conjugational recombination (2, 24).The base pair mutation upstream of mucAB in pGW16

reduces LexA binding. DNA gel shift analyses (7) werecarried out to determine whether the potential LexA proteinrecognition site identified in the wild-type mucAB operon isindeed bound by LexA protein and whether the mutationpresent in pGW16 affects this binding. In this assay, DNAfragments bound by protein demonstrate a reduced migra-tion rate through low-percentage polyacrylamide gels andcan thus be differentiated from unbound fragment. The

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FIG. 4. (A) Partial restriction map of mucAB and flanking regions of the plasmid pKM101. The insertion site of Q1l5::TnS is indicated. Thedirection of transcription of mucAB is indicated by the arrow. The regions essential for pKM101 phenotypes are as follows: MucAB,enhancement of UV and chemical mutagenesis; Rep, region essential for plasmid replication; Bla, ampicillin resistance (see Winans andWalker [46] for a complete map of pKM101). (B) Partial restriction map of plasmid pGW1704. The insertion site of Q116::TnS is indicated.Construction of pGW1704 involved the insertion of a 3.9-kb HpaI fragment into the Hincll gap of pACYC184. The 3.9-kb fragment extendsfrom the HpaI site of the insertion Q1l5::Tn5 to the HpaI-7 site of pKM101. Symbols: , pKM101 DNA; F, TnS DNA; _, vector DNA.(C) Partial restriction map of plasmids pGW1717 and pGW1718. The insertion Q1l6::TnS was recombined into pKM101 and pGW16 asdescribed by Winans et al. (45). Recombinants were screened for their parental Muc phenotypes, and plasmids pKM101 Q116: :TnS and pGW16(116: :TnS were retained for further manipulations. SalI deletion derivatives of pKM101 (116: :TnS and pGW16 (116: :TnS were constructed anddesignated pGW1717 and pGW1718, respectively. pGW1717 and pGW1718 retain the muc, rep, and bla regions of their respective parents.

polymerase chain reaction was used to produce and amplify125-bp fragments of the wild-type and pGW16 upstreamsequences. The fragments contained the putative LexAprotein recognition sequence with approximately 50 bp offlanking sequence to each side. LexA protein efficientlyrecognized and bound the fragment containing wild-typesequence and thereby reduced its mobility through a 5%polyacrylamide gel (Fig. 6a). All of the fragment was appar-ently bound at LexA protein concentrations as low as 6 nM.In contrast, the fragment obtained from pGW16 was notbound by LexA until the protein concentration was in-creased by as much as 30-fold (Fig. 6b). These observationsindicate that the site we identified is indeed a LexA-bindingsite and that the mutation present in pGW16 reduces itsability to bind LexA.

Increased synthesis of pGW16-encoded Muc proteins inmaxicells. To explore the hypothesis that the mutation wehave identified in pGW16 also increases the strength of themucAB promoter, we compared the levels of synthesis of theMucA and MucB proteins from plasmids pGW1717 andpGW1718 in maxicells. Maxicells were prepared from plas-

mid-containing derivatives of the recAJ3 lexA+ strainJC2926. JC2926 was chosen rather than a lexA(Def) strainbecause we have found that pKM101 and its derivatives areunstable in the latter background for reasons that have yet tobe established. However, we have previously shown thatSOS-regulated genes are highly expressed in maxicells de-rived from this lexA+ strain (5, 25), suggesting that LexAeither is degraded during the overnight incubation periodbefore labeling and is not available in maxicells to repressmucAB or else is titrated out by the multiple copies of theplasmid. Furthermore, we have shown that the additionalpresence of a lexA+ plasmid in such maxicells greatlyreduces the expression of plasmid-encoded SOS-regulatedgenes (5, 25).We observed that the amount of MucA and MucB protein

labeled either in the presence or in the absence of LexA wasgreater in maxicells containing the pGW16-derived plasmidthan in maxicells containing the pKM101-derived plasmid(Fig. 7). Quantitation by densitometry indicated that in theabsence of the lexA+ plasmid, the amount of MucA proteinin the extract from maxicells containing the pGW16 deriva-

-60 -50 -40 -30 -205'...CTTGCCAACCTGACCATAACAGCGATACTGTATAAATAAACAGTT ...3'3'...GAACGGTTGGACTGGTATTGTCGCTATGACATATTTATTTGTCAA ... 5'

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FIG. 5. Nucleotide sequence of the 5'-flanking region of mucAB, showing the base pair change carried by plasmid pGW16. Numbering isthat of the mucAB sequence which has been previously determined (26). The +1 nucleotide indicates the start of MucA translation. Thebracket above the sequence indicates homology to the LexA-binding sites of SOS loci. Arrows represent regions of approximate dyadsymmetry. Brackets below the sequence indicate homology to the -35 and -10 regions of E. coli promoters.

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a 1 2 3 4 5 b 1 2 3 4 5 6 7

B-

F-

FIG. 6. Gel shifts assays measuring the binding of LexA proteinto pGW1717 and pGW1718 promoter sequences. DNA fragments(125 bp) containing potential LexA protein recognition sequencesderived from pGW1717 and pGW1718 were end labeled with[y32P]ATP as described in Materials and Methods and incubatedwith increasing concentrations of LexA protein. Binding of LexA tothe fragments was monitored after electrophoresis through 5%polyacrylamide gels and autoradiography. (a) pGW1717-derivedDNA fragment (lane 1) incubated with LexA at protein concentra-tions of 6 nM (lane 2), 30 nM (lane 3), 60 nM (lane 4), and 200 nM(lane 5). (b) pGW1718-derived fragment (lane 1) incubated withLexA at protein concentrations of 3 nM (lane 2), 6 nM (lane 3), 30nM (lane 4), 60 nM (lane 5), 120 nM (lane 6), and 200 nM (lane 7). Fand B indicate the positions of free and LexA protein-bound DNAfragments, respectively.

teins in maxicells containing the pGW16 derivative could, inprinciple, result from either an increased rate of synthesis ofthese proteins or an increase in their stability. The umuDCgene products has been previously shown to be relativelyunstable in maxicell preparations (15). Therefore, we carriedout a pulse-chase experiment to compare the stabilities ofthe pGW1718-encoded Muc proteins with the those of thepGW1717-encoded Muc proteins. Maxicells containingpGW1718 and maxicells containing pGW1717 were labeledfor 15 min with [35S]methionine and chased with cold methi-onine for periods of up to 8 h. There was no apparentdifference between the stabilities of the proteins encoded bythe pGW16 derivative and the stabilities of the proteinsencoded by the pKM101 derivative over this period ofincubation (data not shown). This result indicates that theincreased amount of MucA and MucB proteins in maxicellscontaining the pGW16 derivative reflects an increase in theirrate of synthesis. Because the synthesis of the pGW16-derived Muc proteins is increased relative to that of thepKM101-derived Muc proteins independently of the pres-ence of LexA repressor, these observations are consistentwith the interpretation that pGW16 carries a mutation whichincreases the activity of the mucAB promoter. We haveattempted to assay mucAB promoter activity directly byprimer extension experiments; however, the levels of muc-AB mRNA present in the cells were below our limits ofdetection in these assays.

tive was, on average, increased by a factor of 2.7 over theamount in extracts of maxiceils containing the pKM101derivative. The average increase in the amount of MucBprotein was by a factor of 2.1.The increased amount of labeled MucA and MucB pro-

1 2 3 4 5

MucB-- *

LexA-_

MucA-- .Mt Z it*0I.amI_

FIG. 7. [35S]methionine-labeled proteins produced in maxicellsby plasmids pGW1717 and pGW1718 in the presence and in theabsence of the lexA+ plasmid pSE152. Maxicell extracts wereprepared from plasmid-containing derivatives of JC2926 (recA) afterUV irradiation to 35 J/m2. Proteins were separated by electropho-resis on a 14% polyacrylamide gel and visualized by fluorography.Arrows indicate the positions of the MucA, MucB, and LexAproteins. Lanes: 1, pSE152; 2, pGW1717; 3, pGW1717, pSE152; 4,pGW1718; 5, pGW1718, pSE152.

DISCUSSION

The pKM101 derivative pGW16 is an unusual mutant inthat E. coli strains carrying the plasmid demonstrate higherlevels ofMMS mutagenesis than do strains carrying pKM101but do not demonstrate greater resistance to UV killing. Wehave previously observed a similar phenotype with strainsthat have an elevated dosage of the mucAB operon (27). Datapresented in this report suggest that the cause of this pGW16phenotype is a single-base-pair substitution in the 5' noncod-ing region of the mucAB operon which alters a LexA-bindingsite as well as the -10 region of the putative mucABpromoter. This mutation is therefore of particular interestsince it appears to alter both the operator and promoteractivities of mucAB.

In contrast to its effects in a lexA+ host, pGW16 increasesboth MMS mutagenesis and resistance to UV killing in alexA3(Ind-) host. This phenotype is dominant to the pheno-type conferred by the mucA+B+ plasmid and suggests thatpGW16 carries a mutation which weakens LexA proteinbinding to the mucAB operator. Gel shift assays using afragment containing the putative LexA-binding site of wild-type mucAB confirmed that the LexA protein does indeedrecognize and bind this operator sequence. A fragmentcontaining the pGW16 mutant upstream sequence is notefficiently bound by the LexA protein, indicating that theG * C -3A. T transition mutation alters a base pair requiredfor efficient recognition of the binding site by the LexAprotein. These gel shift assay results are therefore consistentwith there being greater expression of the pGW16-encodedmucAB operon than of a wild-type mucAB operon in alexA3(Ind-) host.Experiments using maxicells indicated that expression of

the pGW16-encoded operon is greater than that of thewild-type mucAB operon under conditions in which LexAprotein seems to be absent. This observation is consistentwith the interpretation that pGW16 carries a mutation whichincreases mucAB promoter activity. As previously de-

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6230 McNALLY ET AL.

scribed, the single-base-pair substitution alters a sequencethat is homologous to the -10 region of E. coli promotersand makes it more closely resemble the consensus sequencefor E. coli promoters. It is therefore likely that the activity ofthe pGW16 mucAB promoter is greater than that of thewild-type mucAB promoter and that this activity also con-tributes to overexpression of the mucA and mucB geneproducts by pGW16.

Several observations indicate that the overproduction ofeither the mucAB or umuDC gene products interferes withaspects of cellular physiology. We have previously shownthat the UV induction of SOS genes is inhibited in cellscarrying a high-copy-number plasmid containing mucAB(16). Furthermore, overexpression of UmuD and UmuC inE. coli results in cold-sensitive growth with a rapid andreversible inhibition of DNA synthesis at the nonpermissivetemperature (15). Our characterizations of the mucAB phe-notypes conferred by pGW16 in a lexA+ background sug-gested initially that the mutation carried by the plasmidaltered specifically its ability to confer resistance to UVkilling and did not alter its ability to enhance mutagenesis. Itis significant that this unique pGW16 phenotype is not theresult of an alteration of the inherent activity of the mucABgene products but rather appears to be due to their overpro-duction. It seems clear that the physiological effects of themucAB and umuDC gene products are strongly influencedby their level of expression. However, the role(s) theseproteins play in mutagenesis and how they might influencenormal cellular metabolism are not yet understood at abiochemical level.Another significant observation is the enhancement of

MMS mutagenesis observed when pGW16 is introduced intoa lexA3(Ind-) host. The lexA3(Ind-) allele effectively pre-vents the induction of SOS-regulated genes. However, themucAB genes of pGW16 would be expressed at elevatedlevels in this host as a consequence of the mutation inpGW16 that alters the LexA-binding site. The finding thatpGW16 substantially increases MMS-induced hisG4 --His'reversion in this host relative to pKM101 suggests that thefunctions that are limiting for mutagenesis are those encodedby mucAB (or umuDC). Any additional SOS-regulated cel-lular factors required for mutagenesis must not be limitingeven in the absence of LexA cleavage.

ACKNOWLEDGMENTS

We are grateful to P. Langer for providing observations of thepGW16 Muc phenotypes, to B. Mitchell for assistance with theDNA sequence determination, and to the members of the laboratoryfor their encouragement and helpful discussions. We thank John W.Little for the gift of LexA protein.

This work was supported in part by Public Health Service grantCA21615 from the National Cancer Institute. K.P.M. was supportedin part by W. R. Grace and was a Swanson fellow. N.E.F. wassupported by a postdoctoral National Research Service award fromthe National Institute of General Medical Sciences.

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