REDUCTION OF SENSITIVITY TO ULTRAVIOLET ...

REDUCTION OF SENSITIVITY TO ULTRAVIOLET EIGHT IN EXCISION-DEFICIENT Escherichia csli STRAIN WP2 U V ~ A EARLER. NESTMANN

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Mutclgertc~rsSec.tion. Envirotrrrtrt~tralant1 Oc-c~uy~r6'otral Toxirologv illvis~on,iJBeprfmrnt r?f ,Yafior~al Heczltbn urrd U'elfare. Tunrmr~'~ Pasture, 0 t t a \ t q a ,Otrtario K I A OE? Mutants with increased resistance to I!V light were isolated from Eschcrdchitr c-oli WP2 revrA. Mc~stof these isolates were designated as true or partial revertants of the r4t9r;4 mutation. However. one class of mutants showed only a moderate increase in rrsistai~ce to UV. The gene rcsponsibla: for this phenotypic change is closely linked to thc lcrc- locus and has been designated the suuA gene (suppressor of uvrA). Des mutants douCs d'une rksistance accrue h 1'U.V. ont 6tP isolis h pareir dc la souche WP2 uvrA d'Esc-hrrbthiu c,oli. La piupart de ces isolats on[ 6tk cl6sign6s cornrne des mutants retcrses tna-aux 0u partiels de la mutation nvr,4 -. Cependant, tine classe cle mutants nc prOsentait qu'un accroissenient 1nodCr6 tie la rksistancc i 19U.V. Le gcnr responsable de cettr: variation phknotypique est itro~tementliC aer Bocus I(ar ct a Ptl. d0signi. ghne sscr4.4 (supprcsseur de uvr.4).

Increased sensitivity of E. coli to UV Iight can result from mutations in genes governing DNA metabolism or ceI1 division (Hanawalt, 1968). Ph~notypicreversion tcs UV resistance in such mutants might be expected to occur either through backramtation or suppression. Previous reports of suppression of UV sensitivity have concerned Rec- and Fal' mutants (Barbour el al.. 1970; Donch eb a!. , 197 8 ). This work reports the suppressic~n of the uvrA- mutation in a derivative of E. coli B/r. A preiiminary report of these results has been published (Nestmann and Hill, 197 I ) .

Materials and Methods Strains Bacterial strains are listed in Table I. Wild type phage TI was used to test for host cell reactivation (HCW),and a derivative of phage Pl kc, able to grow c~nE. cobi B hosts, was used iia transduction experiments. Phage T4 amber mutants B 17, N 133, and NG75, ochre mutants PS28, PS 17 1 , PS205, and PS3 17, and opal mutant PSSM, were provided by S. Person.

Media Bacterial cultures were grown routinely without aeration at 37°C in nutrient broth (NB) containing 0.8% NB powder and 0.5% NaC8. Survival of bacteria and of phage (Adams, 1959) was deternained on N B plates (1.5% agar). Cultures for mapping experiments and for tests for rmonserase suppressors were pre-grown in Luria broth (LB; Luria and Burrows, 1957), and LB broth with glucose (LG), for sukculturing bacteria used in mapping experiments, contained 0.1% glucose. Solid L medium (LB with 1.5% agar: Emratoin, 8969) was used i n experiments with phages $ 1 and T4. Minimal medium (MM), used for mapping experiments, has been described previously (Hill, 1970). To select for Lac' recombinants and transductants, 0.4% lactose (LAC) was used in MM i n place of glucose (MM LAC). Malf 'Based in part o n work done in fulfilltr~entof thc requirements for the h 4 . S ~ degree . at York University. Manuscript received August 2 1 , 1978.

96

E. R. NESTMANN

Bacterial Strains --

U V sensitivity


Strain

Reference

Source

Hill, 1965 Hill. H 965 Hill. I978

Sex

type

Other markers

wild resistant

ma!B trpu/Btrp -mtrlBthr-lc~~c-rri~-izis-~s-inc.xyl-~~nalR-sta'bnutH.s.rrb(origin. . .frp-. ..hi,s.. . .6hv,4-. ..) lau - ochre suppressor rrp -mc~/W-

lavrA uvrA -

Boyer, 1964

H . Boyer

resistant

S. Person This srudy

resistant class IIB

class i class I1 ciass III

C. Fucrst

I&-

c ~ f(origin.. rs .thr-

...ic*u-. ../LU' -. . .) Howard-Flanders eral., 1962 Wcigen and Garcn. 1965 Weigept OI a ! . , I965 Weigen er a / . 1965 Sanbrook, cr d., 1967.

.

S. Beiser P. HowardFlanders S. Person

wild uvrA wild

rkr-ieu -pro -his-thr r g - l a c . ga!-(zr~--.~yl -rnff-fsx'.strr ambers suppressor (Su- l +)

S . Person S . Person S . Person

wild wild wild

amber suppre\sor (Su-2+) ambcr supprclsor (Su-3+) opal suppre\\tar

fhr'^lcu-hio -la(*-.rtrb

transductants were selected on MM agar containing 1% maltose (MM MAL) and required arnino acids (6Opg/ml). In sexduction experiments, Lac+ colonies were obtained on NB-lactose agar (TTC LAC) containing 0.005% triphenyltetrazolium chloride (TTC) and 0.5% lactose. Utilization of rnaltosc was determined by c~bserving both the color of colonial growth OW NB-maltose (TTC MAL) agar, containing 0.005% TTC and 0 to 5 % nnaltose and the presence of growth o n MM MAH, plates.

UV Irradicerlorr The apparatus for UV irradiation, the procedures for determining survival curves, host cell reactivation (HCR), and spot tests, have all been described previously (Hill and Wossi. 1952; Hill and Sirnson, 1961; Hill, 1964; 1965; 1990). The UV flux for this study was 0.94 J/m2/sec. The fluence c~fUV radiation used in spot testing for relative radioresistance was 85 J/m" and in filament tests, tlmences were chosen for 1.5% survival. Bsolarion of UV Rrsfsrasu Derivafive.~ NB plates spread with 108 cells were exposed to a single UV Wuence sf 15 J/ m2,and large colonies formed by survivors were picked for inoculation into NB.

97


REDUCTPON OF U V SENSITIVITY IN E. CQLI

Spot tests of these 2 mi cultures were performed the next day. Derivatives showing increased UV resistance were subjected to a second. single flucnce test, i . e . , survival determined by comparing numbers of colonies arising from after 15 J/m"as irradiated and unirradiated cells. Strains with higher UV resistance than their sensitive UvrA- parents were kept for study. Mating Experm'r?zsnts Hfr? F', and F- strains were grown overnight with aeration. Donor strains were then diluted 8:25 in EG and aerated one h. Mixtures of 5 rnl each of donor and recipient cultures were incubated 60 to 90 min. For F- lac x F- matings. the mixture was shaken (60 cycles/min). After washing, samples of appropriate dilutions were plated on selective media and incubated 48 h. Colonies of recombinants were purified by suspending in saline and streaking on selective rnedia. This procedure was repeated after incubation of the streaks for two days.

Transduc.rion The procedures used for preparing P1 lysaaes and for transduction have been described previously (Euratom, 1969). An overnight culture of the recipient was diluted 1:80 into fresh LG and grown to log phase. At this time, CaCI,-%H,B was added to a final concentration of 2 x 10-W. After the culture was aerated for ten minutes at 37"C, the transducing phage PI was added to the bacteria at a multiplicity of infection 2.0. Phage and bacteria were mixed separately with broth for controls. The transducing and control tubes were slanted for 20 rnin in a 37°C water bath for adsorption. The contents were then mixed with one volume s f warm LB, and the incubation was continued with shaking. After 40 n.lin the tubes were centrifuged for ten rnin at 6000 rpm, and the pellets were suspended in 1/2 volume MgS04-7H2Q ( I x IO-"1. Samples from the tubes were spread on MM LAC, and after two days, the transductants were picked, suspended in MM and streaked can TTC LAC (Euratom, 1969). T'e,~ting for Suppressor Mubarions Dilutions of T4 nonsense mutant stocks were plated with strains to be checked for nonsense suppressors and with their known suppressor hosts as controls. Absence of plaques in the former case and confluent lysis in the latter constituted a negative test for a nonsense suppressor in a test strain (Osborn eb a l . , 1967).

Results Mutants isolated in this study are classified by their resistance to UV (Fig. 1) and by their ability for HCR (Table 11). Class I derivatives ( e . g . , RH5357) show TABLE HI Survival of UV irradiated TI (33.3 .i/m2) --

Moan survival

Host

Derb'varives c$,$uvrA sfrains RH5357 (class I) RH5359 (class IB) RH2332 (class III)

9

cr* (%)

7.0 k 1 . 3 1.8 2 0 . 2 O 01958 004 ----

*c = standard deviation for three expcrirnents.

--


survival curves cc~insidirag with that of WP2 (B/r). The survivals of irradiated TI phage on class I mutants and on the Hcr' controls are not significantly different. Class HI mutants (u.g. ., RH5359) show high UV resistance, but they are slightly UV sensitive compared to WP2. Although survival of irradiated phage TI is 100 tirnes greater when assayed on class II anbatants than on the uvrAs control, HCR ability is not completely restored. Class 11%derivatives ( e . g . ., RH2332 and RH5361) show survival levels higher than that of WB2 M V ~ but A lower than those s f strain WP2, class I or class I1 mutants. They show no improveinen4 in HCR ability. Class 1 derivatives are probably true uvrA6 revertants, and the mutational site was not snapped.

Fig. 1 . Survival of control strains ( @ ) WP2, (V) WP2 rrvrA. and (A)B, and of mutants RH535T-class 1 (sanx as WQ2), (a) RH5359-class 11, ( 0 ) RH2332-class HII, and (5) WH536I -class 111. The points represent averages of three expcrimerats.

(@)


REDUCTION OF UV SENSITBVHTY EN E. COLI

99

Due to nearly complete restoration csf UV resistance (Fig. E) and of HCR ability in class TI strains. an intragenic suppressor mutation was suspected (Gorini and Beskwith, 1966). Since genes uvrA and mall3 are cotransducible at a high frequency (Schwartz, 1966) the ~irlaHB+rnarker of K-12 strain AB1884 (uvrA-) was transduced by phage B1 into class 11 strain WH5359 (mall?-). Spot tests showed that 43% s f the Mal+ transductants were as U V sensitive as AB1886 and that the remainder of the transductants retained class II resistance. P1 grown on one sf the latter transductants (maBB+) was then used to attempt to transfer the class H I allele to UV sensitive WP2 uvrA (rnalB-), and spot bests showed that 89% of the Ma]+ transductants were UV resistant cornpared to the recipient strain WP2 lavaA. These results show that class FI VU resistance probably is due to a nsutation in the lsvrA gene, probably an intragenic suppressor mutation (Gorini and Beckwith, 3966), although a separate gene, also linked to malB, is another possibility. To localize the class TI1 mutation, mating experiments were done. using strains with wild type resistance to UV as donors (HfrH, HB4.2, and 2OOPS) and class 11% strains as recipients (RW5362 and WM5368). These experiments (data not shown) provided evidence that the gene for class HII resistance to UV is more closely linked to the lucs gene than to any raf the other markers in the recipient strains. In the next experiments. phage $1 grown on LVP2 uvrA were used to transduce the genes Bera9 and lac' to class III derivative WH5362. Spot tests show that all 113 Leu+ transductants tested retained the moderate UV resistance of RH5362. On the other hand, 164 of 21 1 Lac+ transductants were UV sensitive, givirag a cotransduction frequency for lac- and the gene for class IHT resistance of 78%. Another possibility examined was that increased resistance of the class II and III mutants is due to nonsense suppression. However, tests for nonsense suppressors (using three amber, four ochre. and one opal, mutants of phage T4) in a11 seven of these bacterial mutant strains (Table I) were negative. The present investigation demonstrates that the UV sensitivity of excisiondeficient E. coli bacteria (UvrA-) can be reduced in at least three different ways. The first pathway involves true reversion to uvrA+ (class I). and the seccmd class probably includes intragenic uvrA suppressor mutations. Class III strains have a suppressor of the eevrA- mutation, designated suuAP, which is ~ B ~ s e llinked y to the lac gene 4 10 min). This may be a direct suppressor of uvrA - whish results in the production of a more functional uvrA repair enzyme (Gorini, 1970). On the other hand, as an indirect suppressor it may partially substitute for the uvrA+ function by opening an alternate pathway, or it may enable the defective U V ~ A -enzyme to work through a change in the cellular environment of class I11 cells (Gorini and Beckwith, 1966). Any suppression of uvaA- might be expected to result in enhanced HCW ability as well as in higher bacterial survival. However, it is known that changes in these characteristics are not always of equal magnitude in E. coli B/r derivatives (Hill, 1964). This may be explained by the different circumstances involved in the repair and replication s f bacterial and phage DNA. Bacterial DNA is repaired and replicated in the absence of phage DNA, whereas phage DNA must be within a bacterium for these processes to occur ir;e vivo. Since irradiated phage TI is not an efficient killing agent (kuria and Delbruck, 194%),bacterial replication continues in competition with the repair and replication of phage DNA. In addition, experinaents in which bacteria or phage were irradiated separately have shown that excision of UV-induced photoproducts from bacterial DNA is about 30 tinaes faster than from phage DNA (Bsyle and Setlow, 1970). Thus, it is not implausible that suppression caf uvrA-

100

E . K. WESTMAWN


would increase bacterial survival with little or no enhancement of phage survival. This preference for b~acterial D N A means that the repair enzymes, which may function in normal DNA replication and editing, are probably localized in the region of bacterial DNA (Boyls and Setlow, 1970).

Acknowledgments This work was partly supported by grant A5737 awarded to the late Dr. Ruth F. Hill from the National Research Council of Canada. I was the recipient of a Graduate Fellowship from the Province of Ontario and a Post-Graduate Scholarship from the National Research Council of Canada. I thank Dr. %. Person for providing nonsense mutants of T4 and their suppressor hosts and Chris Wigham for his help in preparing the manuscript. References Adarns, M . 1959. The bacteriophages. Interscience Publishers. Inc., New B'ork. Barbour, S.. Nagiaiski, H . , Templin. A. and Clark, A.J. 1970. Biochemical and genetic stteclies of recombination proficiency in Escsiaerichin c d i , 11. Recf revertants caused by indirect suppression of Rec- mutations. Proc. Natl. Acad. Sci., U.S.A. 67: 128- 135. Boyer, H . 1966. Conjugation in Escherichicl cnli. I. Bac~eriol.91: L 767- 1772. Boylc, J. M. and Setlow. W.B. 1970. Correlations between host-cell reactivation, ultraviolet reactivation and pyrimidine di~nerexcision in the DNA of bacteriophage. J . Mob. Biol. 51: 13 1- 144. Bonch, J.J., Chung, Y .S., Green, M.H.L., Greenberg, J . and Warren, 6 . 107 1 . Genetic analysis of sul mutants of Escherichin c-oli B. Geaaet. Res. 17: B 85- 193. Eesratorn Course in Molecular Radiokiology. 1969. Orgarzized by the Institut des Sciences et Techniques Nuclcairzs of Sarclay (France) and The Rijksuniversiteit of Leiden (Netherlands). Gorini, L. 1970. Informational suppression. Annu. Rev. Genet. 4: 107- 135. Gorini, L. and Beckwith, J. 1966. Suppression. Annu. Rev. Microbial. 20: 401-422. Hanawalt, P. 1968. Cellular recovery from photochemical damage. Photophysiology 4: 203-25 1 . Hill, R. 8964. Relationship between ultraviolet sensitivity and ability t o propagate a~ltravioiet-irradiated bacteriophage. J . Bacterial. 88: 1283- 1287. Hill, R. 1965. Ultraviolet-induced lethality and reversion to prtrtotrophy in Esc-herichin c,oii strains with normal and reduced dark repair ability. Photochern. Phoeobiol. 4: 563-568. Hill. R. 1970. Location of genes controlling repair of UBr dalnege and mutator activity in Esc%hrrirhicic-oli WP2. Mutat. Res. 9: 341-344. Hill, R. and Rossi, H . 195%. Absence of photoreactivation in 'PI bacteriophage irradiated with ultraviolet in the dry state. Science, 116: 424-425. Hill, R. and Simson, E. 196l. A study of radiosensitivc and radioresistant mutants of Eschc'richia coli strain B.J. Gen. Microbioi. 24: 1- 14. Howard-Flanders, P . , Boyce, W.. Simson, E. and Theriot, L. 1962. A genetic locus in E. caoli K l 2 that controls the reactivation of LIBr-photoproducts associated with thynaine in DNA. Proc. Natl. Acad. Sci. U.S.A. 48: 2109-21 15. Luria, S . ancl Burrows, J . 1957. Hybridization between Escherichiu c ~ l iand Shigel/a. 5 . Bactcriol. '74: 46 1-476. Luria, S . and Delhriick, M . 1942. Interference between inactivated bacterial virus and active virus of the same strain and of a different strain. Arch. Biochem. 1: 207-2 18. Osborn, M., Person, S., Phillips, S. and Funk, F. 1964. A determination of mutagen specificity in bacteria using nonsense mutants of bacteriophage T4. J . Mol. B i d . 26: 437-447. Nestmann, E.R. and Hill, R.F. 1971. Suppression of U V sensitivity in E. c v l i WP2 hcsr-. Can. J . Genet. Cytol. 13: 642. Sanhrook, J., Fan, D. and Brenner, S . 8967. A strong suppressor specific for UGA. Nature (London), 214: 452-453. Sshwartz, M . 1966. Location of the rnaltose A and B loci on the genetic map of Esrhrrickicl c d i . J . BacterioI. 9%:8083-1089. Weigert, M . and Garen, A. 1965. Amino acid substitutions resulting from suppression of nonsense mutations. I. Serine insertion by the Su- l suppressor gene. J. Mol. Biol. 12: 448-455. Weigert, M . , Lanka, E. and Garen. A . 19665. Amino acid substitutions resulting from suppression of nonsense mutations. II. Glutasnine insertion by the Su-2 gene; tyrosine insertion by the Su-3 gene. J. Mol. Biol. 4: 522-527.