LOCI FOR RADIATION SENSITIVITY IN ESCHERICHIA COLI STRAIN Bs-i JOSEPH GREENBERGl
Palo Alto Medical Research Foundation, Palo Alto, California 94301 Received August 2, 1966
TRAIN B,-, is a mutcnt of Escherichia coli strain B, isolated and described by HILLand SIMSON(1961), having the following properties: it is extremely sensitive to ultraviolet (UV) radiation; does not, like parent strain B, form filaments following UV irradiation; is less efficient than strain B at reactivating certain UV irradiated coliphages (HCR-) ; cannot excise UV induced thymine dimers from its DNA and therefore lacks the dark reactivating mechanism of its parent ( SETLOW and CARRIER 1964). It is one of several radiosensitive mutants of strain B which have been isolated by HILLand SIMSON(1961), HILLand FEINER ( 1964), RORSCH,EDELMAN, VANDER KAMP and COHEN(1962), and by EDELMAN and COHEN(1963). RORSCH, We have been attempting to map the loci !which are responsible for radiation sensitivity in strain B and its derivatives. It was established first that strain B itself differs from strain K-12 at a gene linked to the locus for T6 receptor; this was done by using B as a recipient (F-), and K-12 donors, in sexual recombina1964a). By this same method it was found that there was a tions (GREENBERG gene responsible for radiation sensitivity of strain B,-2 which was closely linked to maZB (GREENBERG 1964b) and another gene responsible for radiation sensi1965). HILLand SIMSON tivity of strain B,,, closely linked to his (GREENBERG (1961) guessed that the UV sensitivity of B,-l resulted from two mutations because they could not detect any revertants to UV resistance. This report will show that Bs-l contains two mutant genes, each of which alone can account for radiation sensitivity, and only one of which is responsible for the HCR- property of B,-l. MATERIALS A N D METHODS
Strains: The properties of the donor strains used are shown i n Table 1. The W strains were Stanford University, Palo Alto, Calif.; the AB strains were obtained from DR. J. LEDERBERG, obtained from DR. E. ADELBERG,Yale University, New Haven, Conn.: B and B, strains were obtained from DR.R. HILL,Columbia University, New York, N.Y. Recombinations: The methods for recombination studies were as described by GREENBERG (1964a). Ultrauiolet sensitiuity: Two methods for assay of radiation sensitivity were employed, a long, quantitative method and a rapid, semi-quantitative method. In the rapid method, strains to be assayed were grown overnight at 37°C in tryptone broth containing 1% tryptone, 0.5% NaCl
' This work was carried out under Public Health Service Grant CA 05687-06. Recipient of a Public Health Service Career Development Award. Genetics 5 5 : 19?-201 February 1967.
+ + + + + + I + + + + + + + + + + +
RADIATION RESISTANCE IN
E . coli
and 0.5% yeast extract. Short streaks of culture were applied by capillary tube to tryptone agar containing tryptone 1%, sodium chlorid? 0.5% and BBL (Baltimore Biological Laboratories, Baltimore, Md.) agar 1.5%. Plates were UV-irradiated from a 10 watt General Electrical germicidal lamp with a maximum output a t 2537 A, a t a distance of 51.5 cm from the source. T h e lamp delivered 15.4 ergs/mm?/sec. Plates were incubated approximately 18 hours a t 37°C and examined for the appearance of the streaks. The appearance of streaks of some pertinent strains exposed for several time intervals is shown in Figure 1. Definitive UV survival curves were obtained as follows: cultures of strains to be examined were growii overnight in tryptone broth a t 37°C. Appropriate dilutions in phosphate buffer ( 0 . 0 2 ~containing ) 1% NaCl were spread on tryptone agar plates and irradiated with U\' as ahove. During and subsequent to irradiation plates were kept in subdued light. They were incubated 18 to 24. hours and surviving colonies were counted. The survival curves of several pertinent strains treated in this way are shown in Figure 2. Mulunts: Auxotrophic Lac- and Gal- mutants of donor and recipient strains were obtained by diluting overnight tryptone broth cultures 1: 10 in fresh broth and treating for 30 m i n u t s at 37°C with l-methyl-3-nitro-l-nitrosoguanidine25 to 50 pg/ml. Survivors were plated on tryptone agar or Penassay agar containing triphenyltetrazolium chloride 0.005% and sugar 1%. Auxotrophic mutants were identified by picking surviving colonies onto both tryptone agar and minimal (DM) agar (0.5% glucose; 0.1 % ammonium sulfate; 0.7% dipotassium phosphate; 0.2% monopotassium phosphate; 0.5% sodium citrate; 0.1% magnesium sulfate) and selecting those which failed to grow on minimal agar. These were restreaked and single colonies reisolated before the nutritional requirement was identified. Lac mutants were recognized by the accumulation of dye in the colonies. These were reisolated and tested for growth on minimal agar containing lactose instead of glucose, non-leaky mutants being selected. Gal- mutants were selected in the same manner, but no non-leaky Gal- mutants were found among 15 so isolated. Strain B,-, is naturally Mal- and resistant to valine, whereas all donors were Mal+ and valine sensitive. All streptomycin resistant mutants as well as those resistant to phages were selected from among those occurring spontaneously in the population. Host cell reuc/iuutiom Tests for reactivation of bacteriophage were performed on either TI or T 7 coliphage. Phage (2 x 100) suspended in phosphate-buffered saline (PBS) (1% sodium chloride in 0 . 0 2 ~ phosphate buffer (pH 6.8)) were irradiated with 675 ergs/cm?. Irradiated phage, appropriately diluted, were added to 2.5 ml of semi-solid tryptone agar containing one drop of an overnight culture of test organisms. The semisolid agar was overlaid on tryptone agar and incubated overnight a t 37°C. There were approximately 50 to 100 times as many surviving
FIGURE 1.-The appearance of streaks of cultures of five strains of Escherichia coli following irradiation lot various intervals of time and incubation for 18 hours at 37°C.
following exposure to UV of parental strains used in this study.
plaque-forming units in HCR+ strains as in HCR- strains, All the donors were HCR+ and served as controls; B,-l is HCR- and also served as control. TI was used as test phage unless the recombinant was T i resistant. In this case T7 was used. In crosses involving HfrC a host range mutant of T7 was used, since our stock of HfrC was T7'. T7 was sex-modified: the efficiency of plating on the donor strains was about 10% that of the recipient and the plaques formed on the donors were smaller than those formed on the recipient. Furthermore, most of the recombinants of crosses using, for example, H B 41, behaved like the donor relative to efficiency of plating and plaque size of T7.It was, however, possible to demonstrate HCR in recombinants irrespective of the plating properties of phage T7, provided adjustments were made for differences in efficiency of plating. RESULTS
Many experiments were done involving crosses between HfrC and HfrH and a His- mutant of Bs-l. It was found that, using HfrC as donor, selecting His+ recombinants, no UV resistant recombinants were found among 600 examined. With HfrH as donor, selecting again for the donor his+ marker, only 1.6% of the 700 recombinants examined were UV resistant. One reason one might expect such low frequencies of UV resistant recombinants is that there were two UVs loci in Crosses were performed between HB 33 (BOYER1966) and PAM 11 selecting
RADIATION RESISTANCE IN
TABLE 2 Frequency of donor markers among lac+ str' recombinants of the cross between H B 33 and PAM 11 Frequency
of donor traits
Lac+ str recombinants which were recovered at a frequency of donor cells. UV resistant recombinants constituted 14% of this population (Table 2). These data suggested that one of the postulated UV sensitivity loci of Bs-l might lie in the region between the points of entry of HB 33 and HfrC. However, the frequency of UV resistance was lower than that of proximal donor markers such as TbSand T I s . When a series of auxotrophic derivatives of Bs-l were crossed with HB 33 and prototrophic streptomycin resistant recombinants selected (Table 3 ) , a marked increase in the frequency of UV resistance was observed in some crosses. The cistrons involved in the mutation leading to these auxotrophs was not determined. Nevertheless, UV resistance was more closely linked to markers in the met-arg-ile segment of the coli chromosome than to lac (Table 2), thr or his. However, crosses performed with HB 41 (BOYER1966), for which the arg-met region is proximal to its point of entry, and Bs-l auxotrophs, selecting prototrophic streptomycin resistant recombinants, revealed only 0.2% UV resistant recombinants. This reinforced the idea that there were two genes involved in the UV sensitivity of B8-l and that to obtain UV resistant recombinants it was necessary to incorporate into B,-, the donor region near arg-met and the region proximal to TABLE 3 Frequency of donor markers among recombinants of crosses between HB 33 and Bs-l strr deriuatiues Recipient
aQarg arc a7.r his
(#I) (#2) (#3) (#4)
arg+ strr arg+ strr arg+ strr arg+ strr his+ strr asp+ strr cysf strr iZe+ sir" met+ strr thr+ strr
50 99 96 200
Frequency (%) donor traits
99 30 200 100
32 70 70 32 0 47 3 53 63
The numbers in parentheses represent our number for different isolates. * 93G ergs/mm2 = GO seconds.
TABLE 4 Results of crosses between H B 41 and PAM 12
1a0 100 100
0 2 13
his+ strr lac+ strr * 936 ergs/mm2
Frequency (%) donor traits T1S T6S Lac+ His+
0 13 51
0 15 96
3 33 6
. 6 16
= 60 seconds.
the point of entry of HB 33 but distal to the point of entry of HB 41 and HfrC, presumably the region around lac. Because the met-arg-ile region is distal to the point of entry of HfrH one might expect a low frequency of UV' recombinants in which this donor was used. The idea that there were two genes involved in UV sensitivity of Bs-l was substantiated by experiments summarized in Table 4. In this cross between HB 41 and PAM 12 an increasing frequency of UV resistance was observed in the recombinants as more distal donor markers were selected. Thus, while no UV resistant recombinants were found among 100 selected for Met+, 2% of the His+ recombinants and 13% of the Lac+ recombinants were UV resistant. To test the assumption that one of the two UV sensitivity loci if Bs-l was in the region of metA, 36 Met+, UV sensitive recombinants of the cross in Table 4 were crossed again with HB 41, selecting Lac+ Str recombinants. Twenty-five to 75 recombinants of each backcross were tested for UV resistance. In 11 of the backcrosses the frequency of UV resistance ranged between 0 to 17%. In all but two of the remaining backcrosses, the frequency of UV resistance ranged from 39 to 70%, averaging about 55%. In the two exceptions, the frequency of UV resistance in the backcross was 30%. With these two exceptions, the HB 41 x Bs-l recombinants could be classified into two groups, one of which behaved in the backcross as did the parent Bs-l in the initial cross. The other group (70%) was considered to have acquired the donor UV resistance allele in the metA region in the initial cross. It appeared that there were two UV sensitivity loci in Bs-l one linked about 70% to metA, the other about 55% to lac. Similarly 19 of the Lac+ UV sensitive recombinants of Table 4 were backcrossed to HB 41 selecting Met+ Str. Fifty of each backcross were examined for UV resistance. Again two kinds of HB 41 x Bs-l hybrids were observed: those (47%) in which 0 to 1% of the backcrosses were UV resistant, and those in which the frequency of UV resistance was L 90%. This appeared to confirm the hypothesis that Bs+l differed from B at two loci, one of which was linked about 50% to lac, the other (70 to 90%) to met. It was clear that there were three groups of UV5 hybrids resulting from the cross of HB 41 x B,-, Lac+ Str, those which had acquired UV' genes at neither the metA nor the lac region, those which had acquired the UV' gene in either the metA or the lac region, and those which had acquired both. UV survival and host cell reactivating properties of members of each group were examined. Those
R A D I A T I O N R E S I S T A N C E IN
(13 in number) which had tested UV resistant by the rapid streak method had survival curves indistinguishable from that of strain B and all were HCR+. Those (13 examined) which on backcross to HB 41, selecting Met+ Str recombinants, resembled B,-,, had UV survival curves indistinguishable from Bs-l and all were HCR-. The UV survival curves of those (17 examined) which on backcross with HB 41, selecting Met+ Str recombinants, exhibited a high frequency of UV' recombinants, is shown in Figure 3-B. All were more resistant to UV than B,-,, and at high doses, 2 370 ergs/mm*, more sensitive than strain B. All were HCR+. The UV survival of the Met+ Str recombinants of the HB 41 x BS-, cross in Table 4 which, on backcross to HB 41 selecting Lac+, gave high frequency of UV resistant recombinants, is shown in Figure 3-A. All were somewhat more resistant to UV than B,-,, but significantly less resistant than B or the Lac+ Str recombinants shown in Figure 3-B. All were HCR-. Further confirmation that UV sensitivity of Bs-l resulted from each of two mutations was obtained from the following crosses: HfrH x PAM 12 selecting Lac+ Str recombinants and HfrC x the same recipient, selecting Mal+ Str recombinants. None of the 100 recombinants of each cross was UV resistant by the
UV DOSE lERGS/MM'l
FIGURE 3.-Survival following exposure to UV of hybrids between Hfr HB 41 and Bs.l selected for met (A) and lac (B) . Each dot represents reading of a single recombinant.
rapid method. Twenty-four of the Mal+ Str recombinants of the HfrC x Bs-l cross were crossed with HfrH, selecting 25 Lacf Str recombinants. In 23 (98%) of those crosses the frequency of UV resistant recombinants was Z 35 % averaging 38%. All of the Mal+ recombinants were HCR-. Similarly 31 of the Lacf recombinants of the HfrH X BS-, cross were crossed with HfrC selecting Mal+ Str recombinants, 25 of each being examined f o r U V . Of the 31 Lac+ hybrids, 22 (71 %) had a frequency of UV resistant Mal recombinants 2 SO%, averaging 94%. Those Lac+ Str F, recombinants, which on backcross with HfrC, gave a high frequency of UV resistant recombinants, were all HCR+. It is clear from the data presented that Bs-l relative to B contains two mutant genes effecting radiation sensitivity. One of these genes is linked to metA and maZB. The mutation to UV sensitivity in this gene does not render the mutant HCR-. The other gene associated with the HCR- property is located in the region between the points of entry of HfrC and HB 33, possibly near the gal locus, though no close linkage between it and the particular Gal- mutant employed in these experiments was found. The maZB linked gene may be identical with that in Bs_,, which is also linked to maZB ( GREENBERG 1964b) and which yields a UV sensitive phenotype that is HCR+. In experiments to be described in detail elsewhere, in which the gal locus has been transduced with P I phage from Bs-l to HB 45, a Gal- derivative of B/r (BOYER1966), 24% of the Gal+ transductants were UV sensitive, failing to survive 156 ergs/",. One of the UVs loci of Bs-l is therefore cotransducible with gal and may be identical with the uvrB locus of HOWARD-FLANDERS, BOYCE and THERIOT (1966). Furthermore, one of the B8-, UV sensitivity loci is cotransducible by P I with malB. Over 90% of the mal transductants of BS-, also carried UVI' as an unselected marker. It should be noted that the UV sensitivity of B,-, is greater than that of strains carrying either of its component mutations singly. The phenotypic expression of sensitivity of the two component genes of Bs-l is additive. Additivity of effect has been found in strains containing the genes responsible for HCR- and Rec( HOWARD-FLANDERS and THERIOT 1966). It was not observed in strains containBOYCEand THERIOT 1966). ing more than one HCR- gene (HOWARD-FLANDERS, Nonadditivity of gene effects would appear to reflect very similar, if not identical mechanisms responsible for UV sensitivity. Additivity would suggest differences in mechanisms. During the preparation of this manuscript it came to my attention that DR. ARTHURRORSCH and his colleagues have obtained similar data to those presented here and have submitted these for publication in another journal. (The genetic constitution of the radiation-sensitive mutant Escherichia coli B,-,, Mutation Research, 1966.) I am indebted to MRS.JAN NEALfor her volunteered assistance. SUMMARY
Ultraviolet (UV) sensitivity of strain BS-, is attributable to mutations at two separate loci-one that is linked to maZB and a second that is in the region between the points of entry of HfrC and Hfr HB 33. The mutation to UV sensitivity in
RADIATION RESISTANCE I N
gene 1 is not accompanied by a loss of host cell reactivating (HCR) properties. The mutation to UV sensitivity in gene 2 is accompanied by a loss of HCR. Strain Bs-l is more sensitive to UV than strains carrying mutations in either UV sensitivity gene alone. LITERATURE CITED
BOYER, H., 1966 Conjugation in Escherichia coli. J. Bacteriol. 91: 1767-1772.
J., 1964.a A locus for radiation resistance in Escherichia coli. Genetics 49: 771-778. GREENBERG, __ 1964h A locus for radiation sensitivity in Escherichia coli Genetics 50: 639648. - 1965 Locus for radiation sensitivity in Escherichia coli B,,,. Mutation Res. 2 : 297-303.
HILL, R. F., and E. SIMSON,1961 A study of radiosensitive and radio-resistant mutants of Escherichia coli strain B. J. Gen. Microbiol. 24: 1-14. HILL,R. F., and R. R. FEINER, 1964 Further studies of ultraviolet-sensitive mutants of Escherichia coli strain B.J. Gen. Microhiol. 35: 105-114. HOWARD-FLANDERS, P., R. P. BOYCE,and L. THERIOT, 1966 Three loci in Escherichia coli K-I2 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53 : 1119-1 136. HOWARD-FLANDERS, P., and L. THERIOT, 1966 Mutants of Escherichia coli K-12 defective in DNA repair and in gemtic recombination. Genetics 53: 1137-1150. C. VANDERKAMP,and J. A. CUHEN,1962 Phenotypic and genotypic RORSCH,A., A. EDELMAN, characterization of radiation sensitivity in Escherichia coli B. Biochim. Biophys. Acta 61 : 278-289. RORSCH,A., A. EDELMAN, and J. A. COHEN,1963 The gene-controlled radiation sensitivity in Escherichia coli. Biochim. Biophys. Acta 68 : 263-270. 1964 The disappearance of thymine dimers from DNA: SETLOW,R. B., and W. L. CARRIER, an error-correcting mechanism. Proc. Natl. Acad. Sci. U.S. 51: 226-231.