Mutation Research, 281 (1992) 221-225

221

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 00635

Construction of a umuDC operon substitution mutation in Escherichia coli Roger Woodgate Section on Viruses and Cellular Biology, Building 6, Room 1AI3, National Institute of Child Health and Human Decelopment, Bethesda, MD 20892 (U.S.A.)

(Received 18 October 1991) (Accepted 29 October 1991)

Key~ords: Escherichia coli; SOS mutagenesis; umuDC operon; agtl-aB; Gene replacement

Summary Using a specialized transducing A phage, the u m u D C operon of Escherichia coil was deleted and replaced with the chloramphenicol acetyltransferase gene. The A ( u m u D C ) 5 9 5 : : c a t mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the U m u D , C proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.

Inducible mutagenesis of Escherichia coli requires the products of the u m u D C operon. E. coli strains possessing mutations in either u m u D or u m u C fail to exhibit an increase in mutation frequency normally seen after E. coli is exposed to a variety of D N A replication-inhibiting agents. Only 9 chromosomal u m u mutants have previously been described. Of these, all of the mutants independently isolated by Kato and Shinoura (1977) and Steinborn (1977) are missense mutations (unpublished results). Two insertion mutations have been mapped to the carboxyl-terminal of u m u C (Bagg et al., 1981; Elledge and Walker, 198:3). Recently, an insertion mutation in u m u D has been described (Bailone et al., 1991). Although mutagenesis functions have been compro-

Correspondence: Dr. Roger Woodgate, Section on Viruses and Cellular Biology, Building 6, Room 1A13, National Institute of Child Health and Human Development, Bethesda, MD 20892 (U.S.A.), Tel. (301) 496-6175; FAX (301) 402-0105.

mised, it is conceivable that all of the existing mutants possess partial or full activity of other, as yet uncharacterized U m u D C functions. Similar to E. coli u m u D C mutants, a number of enteric bacteria are not inducible'for mutagenesis. However, many of these enterobacteria possess genes homologous to the E. coli or S. typhimurium u m u operons, and also express a protein homologous to the E. coli U m u D protein (Sedgwick et al., 1991). It has been suggested that these strains may have retained the u m u operon in their genomes through some function (other than inducible mutagenesis) that proved advantageous in evolutionary terms (Sedgwick et al., 1991). Further evidence for additional u m u functions has recently been described by Hiom and Sedgwick who have shown that u m u mutants are defective in restriction alleviation after D N A damage (Hiom et al., 1991). This data, together with the lack of a chromosomal deletion in E. coli of both the u m u D and u m u C genes, has increased speculation that the Umu proteins may perform some

222 as yet uncharacterized function essential for cell survival in addition to their role in mutagenesis. Materials and methods

To test if the Umu proteins are indeed essential, a chromosomal umuDC operon deletion was constructed and conveniently marked with the chloramphenicol acetyltransferase (cat) gene. Although a variety of mechanisms exist to generate deletions, a specialized transducing A vector was chosen that was previously used to construct a deletion of the polA locus in E. coli (Joyce and Grindley, 1984). Use of this phage provides the added advantage that if the desired deletion event is not obtained, progeny phage can be assayed to determine if the recombination event ever occurred, or if it subsequently led to a lethal mutation (Joyce and Grindley, 1984). To construct the substitution in vivo, several in vitro D N A manipulations were necessary. The umuDC operon and flanking sequences were obtained as a 2.8 kb HpaI fragment from p S E l l 7 (Elledge and Walker, 1983) and cloned with EcoRI linkers into the unique EcoRI site of pRW28 (a deletion derivative of pBR322 lacking the single SspI site and the four Eco47III sites). The resultant plasmid, pRW30, was digested with SspI (which cuts 82 bp 5' of the umuD gene and internally in umuC) and Eco47III (which cuts 24 bp 3' of the umuC gene and internally in the umuD gene). Precise removal of the 1.7-kb umuDC operon left ~ 800 bp of flanking D N A 5' to the umuD gene, and ~ 300 bp of flanking sequence 3' to the umuC gene. B a m H I linkers were added to generate a unique B a m H I site at the junction between the flanking sequences. A 1.4-kb B a m H I fragment from pCJ104 containing the cat gene was cloned into the B a m H I site to generate pRW58. A 2.5-kb partial E c o R I fragment containing the cat gene and umuD, C flanking sequences from pRW58 was ligated into the EcoRI phage A vector, Agtl-AB (Cameron et al., 1975), packaged using Gigapack II Gold (Stratagene), and infected into TK603 (Kato and Shinoura, 1977). The structure of the resultant phage, ARW78, was verified by restriction enzyme analysis. Lysogens of ARW78 were obtained by selecting for colonies that were immune to Ach80del9

(Miller, 1972) and screened for chloramphenicol resistance at 30°C. The structure of potential lysogens was verified by Southern blot analysis using a nick-translated 2.8-kb E c o R I fragment from pRW30 containing the entire umu locus and flanking sequences (Maniatis et al., 1982). Results and discussion

The umuDC operon is normally found on a 19-kb chromosomal E c o R I fragment (Shinagawa et al., 1983). Integration of ARW78 via homologous recombination between the flanking umuDC sequences into the 19-kb fragment can result in two theoretical co-integrate structures: One occurring via recombination between upstream sequences, and the other from recombination between downstream sequences (Fig. 1). Since the 5' flanking sequence was larger than the 3' sequence (800 bp vs. 300 bp), it was not surprising that approximately 3 times as many upstream co-integrates than downstream co-integrates were obtained (data not shown). Genomic D N A from two representative co-integrates are shown in Fig. 2. (All the upstream co-integrates tested showed an unexpected extra 1.4-kb band which presumably arose from spontaneous induction of the ARW78 prophage.) Lysogens were cured by plating at 43°C on LB plates containing 1 mM E D T A (Joyce and Grindley, 1984). Colonies were subsequently screened for chloramphenicol resistance and sensitivity to Ach80del9. Excision of the cointegrates can result in two possible structures, one identical to the input, the other the desired replacement event (Fig. 1). Again, as the size of the flanking sequence homology was not equal, an equal number of recombination events was not expected. The upstream co-integrate was predicted to produce mainly wild-type sequences, while the downstream co-integrate was predicted to produce mainly the replacement event. As expected, only 31% of cured bacteria from the upstream co-integrate were chloramphenicol-resistant. Conversely, 78% of the cured downstream co-integrates were chloramphenicol-resistant. To confirm that the umuDC operon had indeed been deleted, genomic D N A was analyzed by Southern blotting (Fig. 2). One clone with the expected restriction pattern (RW82) was chosen

223

R

R

in a 12-fold increase in s p o n t a n e o u s m u t a b i l i t y ( ~ 2-fold g r e a t e r t h a n u m u +) a n d restored ind u c e d mutability to levels g r e a t e r than, or equal to that of the u m u + strain. This confirms that the strain is phenotypically u m u D u m u C - a n d further d e m o n s t r a t e s that both U m u D a n d U m u C p r o t e i n s are actively r e q u i r e d for mutagenesis. T o exclude the possibility that the viability of the d e l e t i o n is not due to a fortuitous second site s u p p r e s s o r m u t a t i o n , the A ( u m u D C ) 5 9 5 : : c a t mutation was t r a n s f e r r e d by g e n e r a l i z e d P1 transd u c t i o n into a variety of genetic b a c k g r o u n d s . C h l o r a m p h e n i c o l - r e s i s t a n t t r a n s d u c t a n t s were assayed either for the n o n - m u t a b l e p h e n o t y p e or loss of the U m u proteins, using a sensitive chemil u m i n e s c e n t w e s t e r n - b l o t t i n g assay that detects c h r o m o s o m a l l y e n c o d e d levels of the U m u pro-

R

D C R

19

~ Integration

R ~37

.

R 11 . .

R .

OR R

D C ~

C ~ m R

R

R

downsrteam

~on D C

~3

R

1 +

4

8

~

19 OR

+

Fig. 1. Theoretical structures obtained when ARW78 integrates via homologous recombination into the 19-kb chromosomal EcoRl fragment containing the umuDC operon and the subsequent excision of the co-integrates to produce the desired substitution event. The closed box represents the 800 bp of flanking sequence 5' to the umuD gene while the open box represents the 300 bp of flanking sequence 3' to the umuC gene. The size of DNA (in kb) expected to hybridize to the 2.8 kb EcoRI fragment from pRW30 containing the umu operon is indicated.

cO p,~

cO

n-

x,,

,~

~--

Cxl Z)

n

or"

"~--- 19

5.3 3.7

for f u r t h e r analysis. This novel u m u s u b s t i t u t i o n m u t a t i o n has b e e n d e s i g n a t e d A ( u m u D C ) 5 9 5 : : cat. "]'he A( u r n u DC ) 5 9 5 : : c a t uvrA6 strain (RW82) was m o d e r a t e l y sensitive to the lethal effects of ultraviolet light, b u t not significantly m o r e t h a n the previously isolated u m u D 4 4 a n d u m u C 3 6 m u t a t i o n s (data not shown). Like the missense m u t a n t s , the s p o n t a n e o u s m u t a b i l i t y of the A u r n u D C strain was r e d u c e d c o m p a r e d to u m u D C +, while i n d u c i b l e m u t a g e n e s i s was totally abolished ( T a b l e 1). I n t r o d u c t i o n of p R W 3 8 (encoding u m u D + u m u C - ) or p R W 4 0 ( e n c o d i n g u m u D - u m u C +) did n o t affect the rate of spont a n e o u s or U V - i n d u c e d mutability. I n t r o d u c t i o n of p R W 3 0 ( u m u D + u m u C + ) , however, resulted

1.4 " ~ ' - - 1.1

Fig. 2. Southern analysis of genomic DNA isolated from ARW78, TK603 (umu +), an upstream co-integrate U, a downstream co-integrate D and the zl(umuDC)595::cat deletion strain RW82.1/zg of genomic DNA was digested with EcoRl, separated on a 0.7% agarose gel and probed with a 2.8-kb nick translated EcoRI fragment from pRW30 containing the umu operon and flanking sequences.

224

teins (Woodgate and Ennis, 1991) (Fig. 3, UmuC not shown). As expected, all (100%) of the chlora m p h e n i c o l resistant strains tested were

recA +

recA +

recA730

lexA +

lexA(Def)

lexA(Def)

+A

+A

+A

AumuDC.

Since

it was

possible

to

transduce

the

a ( u m u D C ) 5 9 5 : : c a t mutation into a variety of genetic backgrounds, it appears that the u m u D C

genes do not encode essential proteins. Whether they have additional functions over and above their roles in mutagenesis and restriction alleviation remains to be resolved. In addition to establishing that the u m u genes are not essential for cell survival, the A u m u D C mutation should prove

TABLE 1 R E S T O R A T I O N OF UV MUTAGENESIS TO A(umuDC)595::cat ucrA6 STRAINS BY PLASMIDS E N C O D I N G umuD A N D / O R umuC Strain

UV a (Jm 2)

M/p b

Sf c

His + induced mutants per 107 survivors

TK603 (umu + )

0 0.5 1.0

33 101 276

1.0 1.0 1.0

24 82

RW82 (ADC595::cat)

0 0.5

6 9

1.0 1.0

0.3

1.0

4

1.0

-

RW82/pRW38 d ( A D C / p U m u D +C - )

0 0.5 1.0

3 4 4

1.0 1.0 0.80

0.2

RW82/pRW40 c ( A D C / p U m u D C +)

0 0.5 1.0

5 5 3

1.0 1.0 0.95

-

RW82/pRW30 ( A D C / p U m u D + C +)

0 0.5 1.0

72 449 594

1.0 1.0 0.94

65 100

The values given are the mean of at least 2 Expts. Cells were plated on minimal media containing 1 /xg/ml histidine; mutation frequencies were calculated as previously described (Sedgwick and Bridges, 1972). a UV, predominantly 254 nm, was given at a fluence of 0.25 Jm 2/sec. b M / p , number of histidine prototrophic revertants per plate. c Surviving fraction of cells after UV-irradiation. d pRW38 was constructed by introducing a frameshift mutation into the umuC gene at the unique Mlul site in pRW30. e pRW40 was constructed by introducing a frameshift mutation into the umuD gene at the unique Ncol site in pRW30.

"~----- U m u D

-~----- U m u D '

Fig. 3. Analysis of wild-type and A(umuDC)595::cat strains using a sensitive western blotting assay. Chromosomal levels of the Umu proteins were detected as previously described (Woodgate and Ennis, 1991). DE1500 (recA + lexA + ) and its umuDC deletion derivative, RW84, were exposed to 1 /zg/ml mitomycin C 2 h prior to cell harvesting to induce U m u D and U m u D ' . Also shown are DE372 [recA + /exA(Def)] and the AumuDC derivative, RW86 and DE274 [recA730 lexA(Def)] and the AumuDC derivative, RW88.

extremely useful in the isolation of novel u m u analogues using techniques based upon DNA or protein hybridization to existing u m u genes or proteins.

Acknowledgements Grateful thanks are extended to Cathy Joyce for generously supplying bacteriophages and plasmids necessary to construct the A u m u D C substitution mutation; Don Ennis for bacterial strains and comments on the manuscript; Arthur S. Levine for helpful suggestions and comments on the manuscript; Franca Pompetti for help and advice with the Southern blots; and Alvaro Puga for helpful suggestions.

References Bagg, A., C.J. Kenyon and G.C. Walker (1981) Inducibility of a gene product required for UV and chemical mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5749-5753. Bailone, A., S. Sommer, J. Knezevic and R. Devoret (1991) Substitution of U m u D ' for U m u D does not affect SOS mutagenesis, Biochimie, 73, 471-478. Cameron, J.R., S.M. Panasenko, I.R. Lehman and R.W. Davis (1975) In vitro construction of bacteriophage A carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase Proc. Natl. Acad. Sci. (U.S.A)., 72, 3416-3420.

225 Ellectge, S.J., and G.C. Walker (1983) Proteins required for ultraviolet light and chemical mutagenesis, Identification of the products of the umuC locus of Escherichia coli, J. Mol. Biol., 164, 175-192. Hiom, K., S.M. Thomas and S.G. Sedgwick (1991) Different mechanisms for SOS induced alleviation of DNA restriction in Escherichia coli, Biochimie, 73, 399-406. Joyce, C.M., and N.D.F. Grindley (1984) Method for determining whether a gene of Escherichia coli is essential: application to the polA gene, J. Bacteriol., 158, 636-643. Kato, T., and Y. Shinoura (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light, Mol. Gen. Genet., 156, 121-131. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Miller, J.H. (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 295.

Sedgwick, S.G., and B.A. Bridges (1972) Survival, mutation and capacity to repair single strand DNA breaks after gamma irradiation in different exr strains of Escherichia coli, Mol. Gen.'Genet,, 119, 93-102. Sedgwick, S.G., C. Ho and R. Woodgate (1991) Mutagenic DNA repair in Enterobacteria, J. Bacteriol., 173, 56045611. Shinagawa, H., T. Kato, T. Ise, K. Makino and A. Nakata (1983) Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli Gene, 23, 167-174. Steinborn, G. (1978) Uvm mutants of Escherichia coli KI2 deficient in UV mutagenesis, I. Isolation of ucm mutants and their phenotypical characterization in DNA repair and mutagenesis, Mol, Gen. Genet., 165, 87 93. Woodgate, R., and D.G. Ennis (1991) Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage, Mol. Gen. Genet., 229, 10-16. Communicated by R.P.P. Fuchs