GENETICS OF THE TEMPERATURE-DEPENDENT AND -SENSITIVE REVERTANTS OBTAINED FROM THREONINE-REQUIRING MUTANTS OF BACILLUS SUBTILIS

Transcription

1 J. Gen. Appl. Microbiol., 15, (1969) GENETICS OF THE TEMPERATURE-DEPENDENT AND -SENSITIVE REVERTANTS OBTAINED FROM THREONINE-REQUIRING MUTANTS OF BACILLUS SUBTILIS OSAMU KANAMITSU1 AND YONOSUKE IKEDA The Institute of Applied Microbiology, University of Tokyo, Tokyo (Received May 28, 1969) Revertants obtained from three threonine-requiring mutants of Bacillus subtilis were studied. The revertants from mutants 347 and 350 did not form normal colonies at 37 while they did at 50 (temperature-dependent), and the revertants from mutant 530 did not form colonies at 50 while they did at 37 (temperature-sensitive). By genetic analysis, one reversion observed in 350 was attributed to a mutation occurring in the same cistron, and one reversion observed in 530 was attributed to a suppressor mutation occurring at a map position of Mechanism of reversion was discussed. It was described by MAAS and DAVIS (1) that some of the prototrophic revertants obtained from a pantothenate-requiring mutant of.escherichia coli produced a heat-labile protein with low enzymatic activity. It was thought that the partial restoration of the enzymatic activity might be caused by a mutation occurring in the pan cistron or in a separate cistron. This paper describes temperature-dependent and -sensitive revertants obtained from threonine-requiring mutants of Bacillus subtilis. Two threoninerequiring mutants yielded revertants that grew at 50 but not at 37, and one threonine-requiring mutant yielded revertants that grew at 37 but not at 50 in threonine-free medium. It was evidenced by genetic analysis that one of the temperature-dependent revertants was produced by a mutation occurring in the threonine cistron and one of the temperature-sensitive revertants was produced by a suppressor mutation occurring in a separate cistron. MATERIALS AND METHODS Strains. A wild type strain of Bacillus subtilis Marburg and auxotrophic mutants derived from this strain were used. Strain 160 (trp) and strain 1 Entrusted Research Fellow from the Technical Research Laboratory, Asahi Chemical Industry Co., Ltd., Itabashi-ku, Tokyo, Japan. 463

2 464 KANAMITSU AND IKEDA VOL. 15 Mu8u5u5 (leu thr met) were supplied by Dr. D. M. BONNER and Dr. N. SUEOKA, respectively. Strain 6160 (ade leu met trp) was a gift from Dr. J. NUKUSHINA. Threonine-requiring mutants, 347, 350, and 530, were derived from strain 160 by treatment with nitrosoguanidine (N-methyl-N'-nitro-Nnitrosoguanidine). Media. Spizizen's minimal medium (2) containing (per liter) glucose 5.0 g, KH2PO g, KOH 2.26 g, sodium citrate.2h g, (NH4)2SO4 1.0 g, MgSO4.7H2O 0.2 g, L-tryptophan g, L-arginine HC10.05 g, and agar (Difco) 15 g was used for the experiments of reversion and transformation. When L-threonine was to be supplemented, it was used in an amount of 0.05 g/liter. Liquid growth medium containing (per liter) glucose 5 g, KH2PO g, KOH 2.26 g, sodium citrate 2H2O 0.5 g, (NH4)2SO4 1.0 g, MgCl2.6H2O 0.5 g, MnCl2 4H2O g, calcium acetate H2O 0.02 g, sodium L-glutamate 2.0 g, and L- tryptophan 0.05 g was used for the experiments of growth pattern. Treatment with nitrosoguanidine. Cells harvested from an early logarithmic growth phase in nutrient broth were washed twice with a phosphate buffer (0.1 M, ph 6.0) supplemented with 2 X 10_g M MgSO4 and incubated at 37 with shaking for min in the phosphate buffer containing pg/ml of nitrosoguanidine. The cells were washed with the same buffer and suspended in nutrient broth. Plating onto nutrient agar was carried out after incubation at 37 for 5 hr. Auxotrophic mutants were selected by the replica method. Transformation procedures. DNA was prepared by the method of SAITO and MIURA (3). Other procedures were the same as those described by TANOOKA and SAKAKIBARA (4). Map position of a gene on the chromosome was determined by the marker frequency method described by YOSHIKAWA and SUEOKA (5). The trplso was used as the terminal marker instead of the met5. The marker frequency of ades to trplso was (Table 5). RESULTS Characterization of the revertants obtained from thr strains A threonine-requiring mutant cultured in nutrient broth was plated onto minimal medium devoid of threonine, and the plates were incubated at 37 and 50. The number of colonies which appeared on the plates is recorded in Table 1 (a). Some of the revertants might be of the same origin. Mutant 347 and mutant 350 yielded revertants only on the plates incubated at 50 and mutant 530 yielded revertants only on the plates incubated at 37. Growth character of the revertants was tested on minimum medium by picking up 20 colonies from each (Table 1 (b)). All the revertants from 347 and 350 formed colonies at 50, but not at 37. Out of 20 revertants from 530, 19 did not form colonies at 50, while all of them did at 37. All the revertants from 347, 350, and 530 formed colonies both at 37 and 50 on threonine-supplemented medium. Hereafter, the revertants from 347 and 350

3 1969 Revertants of thr Auxotrophs in B. subtilis 465 Table 1. Appearance of from temperature-dependent threonine-requiring and -sensitive mutants. revertants will be called temperature-dependent and those from 530 will be called temperature-sensitive with regard to threonine requirement. Fig. l shows growth of the temperature-sensitive and -dependent revertants in a liquid medium. It is suggested that the dependence of a revertant from 350 on temperature is not so strict as in the solid medium as far as the growth is estimated from the change of optical density at 610 m,u. Genetic analysis of the revertants If the revertants are true (intracistronic) revertants, the thrtd and thrts genes may be transformed to thr+ by DNA from a wild type strain and the transformed cells may work as the donor of thr+. On the other hand, if the revertants are suppressor revertants such as thr su+, the thr may be transformed to thr+ by DNA from a wild type strain and the transformed cells may work as the donor of thr~ and su+. It is expected, therefore, that a thr strain may be transformed to thr+ by DNA of the former type (thr+) and to both thr+ and thr su+ by DNA of the latter type (thr+ su+). The thr+ transformants will grow both at 37 and 50, and the thr sup transformants will grow either at 37 or 50. Table 2 shows results of the transformation of thr markers in strains 347, 350, and 530 by DNA's from phenotypic thr+ strains. These results suggest that 347-HR22, 347-HR24, 350-HR21, 350-HR23, and 530-R13 may be intracistronic revertants and 530-R2 may be a suppressor revertant.

4 466 KANAMITSU AND IKEDA VOL. 15 Fig. 1. Growth of threonine auxotrophs and their revertants at 37 and 48 in threonine-free medium. Growth at 37 (-0-) and at 48 (-- --) in threonine-free growth medium described in MATERIALS AND METHODS. 350 and 530: Threonine-requiring mutants. 350-HR21: A revertant from R2: A revertant from 530. Mutual crosses of the various thr mutants As shown in Table 3, the thr in 350 was transformed to thr+ by DNA from 530 and the thr in 530 was transformed to thr+ by DNA from 350. As the thr+/trp+ ratios were significantly lower than those by DNA from a wild type strain, the two loci, thr35o and thr53o, might be located close to each other on the chromosome. It was also shown by these experiments that the thrtd in 350-HR21

5 1969 Revertants of thr Auxotrophs in B. subtilis 467 Table 2. Growth response to threonine-requiring mutants by temperature treatment with of the transformants obtained DNA from phenotypic thr+ from strains. could be introduced into 530 and 350. Progeny from 530 that had received Su+530-R2 were temperature-sensitive, but those from 350 that might have received Su+530-R2 Were either temperature-insensitive or unrescued. It was thought, therefore, that the co-existence of thr53o and Su+530-R2 genes might be necessary for the expression of temperature-sensitive character. Derivation of thr transformants with DNA of 530-R2 If the genotype of 530-R2 is thr su+, the thr must be introduced into the chromosome of recipient strain by transformation. The transformation was carried out by using strain 6160 (ade leu met trp) as the recipient. The cells were exposed to high concentrations of DNA from 530-R2 so that each recipient cell might receive more than two DNA pieces, and threonine-requiring progeny were selected among the ade" transformants. As shown in Table 4, the ade+ thr transformants were really recovered from the lots treated with DNA from 530-R2 and their ratios to the total ade+ were almost equal to those in the lots treated with DNA from 530. Map positions of Su+53o_R2, thr350, and thr53o Map position of su+ in 530-R2 was studied by the marker frequency method devised by YOSHIKAWA and SUEOKA (5). From the data in Table 5, the position is estimated to be 0.64 from the origin. The map positions of thr350 and thr53o were studied by using mutant Mu8u5u5 (thr5) as a reference. The thr5 in the reference had been mapped by YOSHIKAWA and SUEOKA. The results in Table 6 suggest that the thr350 and thr53o are

6 468 KANAMITSU AND IKEDA VOL. 15 Table 3. Results of Donor strains were prepared from the corresponding trp strains by transform- located closely to the thr5. As 350 and Mu8u5u5 had the activity of homoserine kinase [ATP : L-homoserine o-phosphotransferase, EC ] and 530 was defective in it, the latter may belong to another class of the formers. Activity of the enzyme was measured by the method of WORMsER and PARDEE (6). Fig. 2 shows the presumed map positions of the four genes studied. DISCUSSION According to GORINI and BECKWITH (7), the suppressor mutations are classified into six categories. (1) Opening of an alternative pathway, (2) substitution of the function of the product of the first gene, (3) alteration of the cytoplasmic conditions which affect the structure of the finished product of the first mutated gene, (4) introduction of a second alteration within the

7 1969 Revertants of thr Auxotrophs in B. subtilis 469 mutual ation crosses. of trp + same peptide chain, (5) reintroduction of the correct code-meaning, by frame shift, within the same cistron, and (6) alteration of the factors controlling the mechanism of transfer of genetic information from DNA to protein. The reversions observed in 347 and 350 are due to the intracistronic mutations. The revertants might be produced by category (4) stated above. One of the histidine-requiring, temperature-dependent mutants of E, coli isolated by O'DONOVAN and INGRAAM (8) has been reported to produce an altered phosphoribosyl-atp pyrophosphorylase which is more sensitive to feedback inhibition at 20 than at 37. The suppressor mutation which occurred in 530-R2 may be inferred by category 1, 2, 3, or 6. Among these categories, alteration of the structure of transfer RNA or ribosome is considered to occur most frequently. On the preposition that the su~530_12 may work through this mechanism, isolation of the mutants of bacteriophage M2 capable of being rescued by the su~53o-r2

8 470 KANAMITSU AND IKEDA VOL. 15 Table 4. Derivation of thr transformants from a wild type strain by DNA's from 530 and 530-R2. Strain 6160 (ade leu met trp thr+) was used as the recipient strain. The ade~ thr transformants obtained did not grow on adenine-supplemented minimum medium either at 37 or 50, and they might be true thr. Table 5. Determination of map position of su "53o-R2 Map position of su+530-r2 was determined according to the marker frequency method. DNA's were prepared from the cells of 530-R2-F15 (thr Su+530-R2 trp~) grown to the stage of an early log phase and a stationary phase. For standardization, 6160 (ades leu met trpiso) and (trpis0 thr35o) were used as recipients. The data were derived on the assumption that map position of trpis0 is was attempted. All the mutants tested (approx. one thousand) grew on both su and su+ cells. A supressor-sensitive mutant of bacteriophage SPO1, sus 8, which was isolated by OKUBO and YANAGIDA (9) (kindly given by Dr. S. OKUBO) was also tested on 530-R2. The phage was not propagated on 530- R2. Possibility of a suppressor of nonsense type might be poor in su+530-rti. Other mechanisms should be studied hereafter. The reason why mutants 347, 350, and 530 do not yield a large number of normal type revertants and the properties of the threonine-synthesizing enzymes in the revertants are the subjects for future study.

9 1969 Revertants of thr Auxotrophs in B. subtilis 471 the the Table 6. Determination of map positions of thr350 and thr530. Mutant Mu8u5u5 (leu5 thr5 met5) was used as the recipient. Map position of thr5 was reported to be 0.26 (YOSHIKAWA and SUEOKA). Donor strains were same as in Table 3. Fig. 2. Map positions of the thr5i thr350, thr530 and ]I2 loci on the chromosome of B. subtilis. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) W.K. MAAS and B.D. DAVIS, Proc. Natl. Acad. Sci. U.S., 38, 785 (1952). J. SPIZIZEN, Proc. Natl. Acad. Sci. U.S., 44, 1072 (1958). H. SAITO and K. MIURA, Biochim. Biophys. Acta, 72, 619 (1963). H. TANOOKA and Y. SAKAKIBARA, Biochim. Biophys. Acta, 155, 130 (1968). H. YOSHIKAWA and N. SUEOKA, Proc. Natl. Acad. Sci. U.S., 49, 559 (1963). E.H. WORMSER and A.B. PARDEE, Arch. Biophys. Biochem., 78, 416 (1958). L. GORINI and JR. BECKWITH, In Annual Review of Microbiology, ed. by CE. Clifton, Ann. Reviews Inc., Palo Alto, Calif. (1966), Vol. 20, p GA. O'DONOVAN and J.L. INGRAHAM, Proc. Natl. Acad. Sci. U.S., 54, 451 (1965). S. OKUBO and T. YANAGIDA, J. Bacteriol., 95, 1187 (1968).