Molec. gen. Genet. 149, 201-210 (1976) © by Springer-Verlag 1976
Revertants from RNase III Negative Strains of Escherichia coli David Apirion, Jeff Neff, and Ned Watson Washington University Medical School, Department of Microbiology and Immunology, Division of Biology and Biomedical Sciences, St. Louis, Missouri 63110, USA
Summary. E. coli strains carrying the r n c - 1 0 5 allele do not show any level of R N a s e III in extracts, grow slower than rnc + strains at temperatures up to 45 ° C and fail to grow at 45 ° C. Revertants which can grow at 45 ° C were isolated. The vast majority of them still do not grow as fast as rnc + strains and did not regain R N a s e III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of R N a s e III. They do not harbor the r n c - 1 0 5 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate r n c + p u r revertants from an r n c - p u r - strain with relative ease. They behaved exactly like the true rnc ÷ revertants isolated from r n c - strains at 45 ° C. A merodiploid strain which contains the rnc + gene on an episome behaves exactly like an rnc + strain with respect to growth and R N A metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc + strain; thus the level of R N a s e III is not limiting in the cell. The m c - strains show a characteristic pattern of transitory molecules, related to r R N A , 30S, 25S, " p 2 3 " and 18S, which are not observed in rnc ÷ strains. This pattern is unchanged in r n c - strains and in the revertants which are still lacking RNase III, regardless of the temperature in which R N A synthesis was examined (30 ° to 45 ° C). On the other hand, in the mc + strains as well as in the true revertants and the r n c + / r n c - merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase I I I - strains are due to pleiotropic effects of the r n c - 1 0 5 allele, and that the enzyme R N a s e I I I is not essential for the viability of the E. coli cell.
Introduction E. coli strains which carry the r n c - 1 0 5 allele do not
show a significant level of R N a s e III activity in cell
extracts (Kindler, Keil and Hofschneider, 1973; Apirion and Watson, 1974, 1975a, b; Apirion, Neil and Watson, 1976). The original strain AB30 I- 105 isolated by Kindler et al., besides being RNase I I I - , contains a large number of mutations (Apirion and Watson, 1974, 1975b). Therefore when it was shown that strain AB301-105 has an abnormal r R N A metabolism (Dunn and Studier, 1973; Nikolaev et al., 1973) it became necessary to establish to what extent the mutation affecting RNase III is also altering r R N A synthesis. While previous studies (Apirion, 1974; Apirion, Gegenheimer and Watson, 1975; Apirion, Nell and Watson, 1976) indicated that the r n c - 1 0 5 mutation is causing all these pleiotropic effects, they could not rule out the possibility that another mutation very closely linked to r n c - 1 0 5 might be involved in some of these effects. Such a possibility could have been entertained with some credence since previous studies showed that the original r n c - 1 0 5 strains carried a second mutation which did not affect the level of RNase III, but conferred inability to grow at 4 5 ° C (Apirion and Watson, 1975 b) and affected R N A metabolism, probably the maturation of 17S to 16S r R N A (Gegenheimer and Apirion, unpublished observations). In the studies reported here we describe the isolation of revertants and their properties. We show that indeed the r n c - 1 0 5 mutation is causing all the pleiotropic effects found in R N a s e I I I - strains. These studies also suggest that strains carrying the r n c - 1 0 5 allele do not contain RNase III and therefore this enzyme is not essential for the viability of the E. coli cell.
Material and Methods Strains. All strains used are described in Table 1. Media were described previously (Apirion, 1966; Lenncne and
Apirion, 197i), except for low phosphate rich medium which was prepared as follows: Eight grams of nutrient broth and 8 grams of casamino acids were dissolved in 800 ml of water and the pH was adjusted to 10.5 with concentrated NH4OH. Sixty six ml of 1M MgC12 was added dropwise with stirring over a period of 15 minutes. The mixture was then allowed to stir for an additional 30 minutes. The precipitate formed was collected on a Whatman
202
D. Apirion et al. : Revertants from RNase III- Strains
Table 1. Strains used
Strain
Markers
Source
PA3306
F - t h i - i pur I66 argH1 nadB4 lac Y1 g a l - 6 m a l A 1
Lavalle"
N2076 N2077 N2078 N2080 N2082 N2084 N2087 N2088 N2089 N2090 N2097 N2100 N2403 N2416 N2419 N3705
a b c d
2R x y l - 7 a r a - t 3 m t l - 2 str-9 t o n A 2 ? 2 supE44 R F See PA3306 a b o v e , pur + ranA + rnc + F - See PA3306 above, p u t + rne-105 ranA + F - See PA3306 above, pur + nad+ rnc-105 ranA + F rne-105 spontaneous revertant F - rne-105 spontaneous revertant F See PA3306 above, pur + n a d + rne-I05 ranA + F - rne-105 spontaneous revertant
F - rne + spontaneous revertant F - lae + derived from strain N2076 F - lae + derived from strain N2077 F - mal + a spontaneous derivative F See PA3306 above, p u r l nad+ rnc-I05 ranA + F - rnc-105 spontaneous revertant F - rnc + spontaneous revertant F - rnc + spontaneous revertant F' chromosome like in strain N2100 episome F' 142 tyr s u p N (Low, 1972)
N2066~PA3306 b c N2066--+PA3306 N2066~PA3306 from strain N2077 d from strain N2078 d N2066~PA3306 from strain N2084 d from strain N2084 d D10~N2076 d D10--,N2077 a from strain N2090 a N2084~PA3306 d from strain N2077d from strain N2077 d from strain N2100 a KLF42/KL253 x N2100 d
The n o n N strains were supplied by B.J. Bachmann, Yale University, Coli Genetic Stock Center Denotes a transduction; arrow leads from donor to recipient Apirion and Watson, 1975b This paper
No. 5 filter paper. The filtrate was autoclaved for 3 minutes. The resulting solution was filtered again on W h a t m a n No. 5 filter paper, the pH was adjusted to 7.0 with concentrated HC1, the final volume was adjusted to 1.0 liter, and the medium was autoclaved. Protein Determination. This was carried out according to the procedure of Ehresmann, Imbualt and Weil (1973). All other techniques used were previously described. For P1 mediated transduction see, Apirion and Watson (1975), Lennette and Apirion (1971). For assaying RNase III see Apirion and Watson (1974) Apirion et al. (1976). For labeling cells with 32Pi, preparation of extracts, and electrophoresis in polyacrylamide gels, see Gegenheimer and Apirion (1975), Kaplan and Apirion (1975) and Meyhack et al. (1973).
Results I s o l a t i o n o f Ts + R e v e r t a n t s . a) B y G r o w t h a t 4 5 ° C. We reported earlier (Apirion and Watson, 1974; Apirion and Watson, 1975b) that strains which carry the r n c - 1 0 5 allele cannot grow at 45 ° C. Therefore we isolated Ts + revertants by plating r n c - 1 0 5 cells on rich medium plates and incubating them at 45 ° C. Numerous colonies appeared which regained the capacity to grow at 45 ° C. On purifying and retesting these colonies, they behaved as revertants, since after numerous passages (at least 5) at 37 ° C they did not alter their capacity to grow at 45 ° C. Ts + revertants were isolated from a number of strains carrying the r n c - 1 0 5 allele. All such strains revert with a similar frequency, around 10 -5 to 10 6). In order to measure the reversion rate a number of experiments were carried out, one
of which is described below. Fifty cultures were started from different clones of strain N2077, and about 105 cells from each culture were plated on a rich medium plate at 45 ° C. F r o m the 50 samples, 14 had not Ts ÷ revertants. The reversion rate calculated from the zero class (Luria and Delbruck, 1943) was 3 , 4 x 1 0 -6, whereas the reversion frequency was 3 x 10-6 The revertants did not grow as well as rnc ÷ strains at all tested temperatures. This was first observed in replication tests. To make the distinction easier, colonies were streaked out at 45 ° C on rich medium plates. Such tests showed rather clearly that the single colonies from revertant cells, observed in a streak, were smaller in size than colonies from rnc ÷ strains. (A number of rnc ÷ strains were tested, including N2076, the rnc ÷ isogenic partner of the r n c - strain N2077.) Measuring the doubling times of the revertants showed that they do grow somewhat faster than the r n c - parental strains but slower than rnc + strains (Table 2). Further analysis (see below) showed that these revertants lack RNase III. In order to find out if r n c - 1 0 5 is likely to be a point mutation, we tried to isolate true revertants. This was accomplished by plating large numbers of r n c - cells from independent clones on rich medium plates at 45 ° C and picking out from each clone four large revertants. After retesting these large revertants by replication and streak tests, the more promising candidates were assayed for R N a s e III, and some clearly regain RNase III activity (see below). We tried
D. Apirion et al. : Revertants from RNase I I I - Strains
203
Table 2. Doubling times of various strains in rich medium at different temperatures (min)
Table 3. A reconstitution experiment
RNase III
37 C
45 C
rnc-105
No. of colonies observed
No. of colonies tested
Strain N2076 N2089 N2077 N2084 N2100 N2080 a N2087 a N2403" N2088" N2416 a N2419 a N3705 (rnc+/rnc-)
+ + -
45 46 71 73 77 54 63 61 48 47 49 47
56 61 oo oo co 71 75 77 52 53 52 58
cells plated
Large
Large
No. of
+ + +
+
Revertant strains
to isolate such revertants from three different rncstrains, and succeeded in doing so with all of them (for further analysis of these Ts + revertants, see below). b) By Growth on Ademlne Analogues. In order to elimi-
nate the numerous revertants which did not regain RNase III, we resorted to adenine analogues. We found that a p u r - r n c - cell is disadvantaged when adenine is replaced with limited concentrations of other purine bases. We tested a number of those by auxanographic techniques (Pontecorvo, 1949) and found that a medium containing 2 gg/ml of either xanthine or 6-methylaminopurine (MAP) as a substitute for adenine will allow rnc+purl - cells to grow better than r n c - p u r I - cells. In order to ensure that we would be able to use this technique, we ran reconstitution experiments plating about 80 cells per plate of the p u r I - r n c + strain PA3306 with increasing numbers of the p u r I - r n c strain N2100, from 0 to 107 cells per plate (Table 3). We found that by using xanthine it was possible to recover at least 75% of the PA3306 cells when cells of the N2100 strain did not exceed 105 cells per plate (Table 3). When the N2100 cells were plated 106 per plate, about 50% recovery of PA3306 cells was obtained, but no rnc + cells were recovered when the number of N2100 cells were increased to 107 per plate. The background cells, N2100, were observed as smaller colonies at all concentrations tested, becoming relatively smaller with the increase in cell concentration. This was verified by picking out from the test plates large and small colonies, purifying them and testing them. As excepted, all the originally small colonies were Ys- Nic + (as is N2100) and all the large colonies were Ts + Nic- (as is PA3306). Moreover, five colonies from each of these groups were assayed for RNase III and
Small
Small
FHC +
I'~lC-
r~c +
r~c-
0 103 104 l0 s 106 107
83 78 84 68 32 0
0 320 1700 a a a
10 i0 10 8 l0
0 0 0 2 8
0 0 0 10 0 10 0 8 0 8 Not tested
0 103 104 l05 l06 l07
82 98 97 73 18 10
0 2300 ~ 3000 ~6000 a a
10 8 6 7 15 10
0 2 4 3 0 0
0 0 0 10 0 8 0 10 Not tested Not tested
A
A background of limited growth forming a thin lawn of cells 0.1 ml of a culture of strain PA3306 (rnc+purI66 nadB4) containing about ninety cells, and 0.1 ml of a culture from strain N2100 (rnc-105 purI66 had+) containing various amounts of cells as indicated, were plated on minimal medium plates containing thiamine nicotinic acid and arginine. To one set of plates xantine (A) and to another MAP (B) were added (2 gg/ml). The first set of plates were incubated for four days and the second for six days. A number of large and small colonies were picked up, purified and characterized. Colonies whichgrew slower and were Nad + and Ts were classified as N2100 (rnc-) while colonies which were fast growers, N a d - and Ts + were classified as PA3306 (rnc+)
again all the Ts- colonies were RNase I I I - , while all the Ts + colonies were RNase III +. When MAP was used in a reconstitution experiment, we found it necessary to incubate the plates somewhat longer, 6 rather than 4 days, and that at lower to medium concentrations of rnc- cells (103-105) the distinction between the rnc- and rnc ÷ cells is not clear cut. However, at high concentrations of rnc- cells, 106 and 107 cells per plate, the recovery was only around 20 and 15% respectively (Table 3), but all the distinctly large colonies tested as PA3306 cells (tested as above). To isolate RNase III+ revertants, we plated strain N2100 on plates containing 2 gg/ml of either xanthine or MAP (no adenine) and plates were incubated at 37 ° C. Colonies appeared on both types of plates. Twenty colonies were tested in some detail, 6 which appeared on the xanthine plates and 14 which appeared on the MAP plates. All the six colonies isolated from the xanthine plates resulted from reversion to Pur + and remained T s - . F r o m the 14 isolated from the MAP plates only 1 was a Pur+Ts - revertant, 4 were P u r - T s ÷ and the other nine remained P u r - T s - but became mucoid (which helped them to establish distinct colonies on the background of the non-mucoid
204
D. Apirion et al. : Revertants from RNase III- Strains
cells). In this particular experiment, the reversion frequency to Ts + was 2 x 10- 6. These four strains contain normal levels of R N a s e III (see below).
100
C h a r a c t e r i z a t i o n o f R e v e r t a n t S t r a i n s . A number of revertant strains were chracterized for growth, for the ability to be used as host for various phages, and for levels of the RNase III enzyme. With respect to the level of enzyme, we found invariably that all the slow growing revertants remain RNase I I I - exactly as their parental strains. (We tested during the past two years about 280 independently isolated revertants.) On the other hand, all the normally growing revertants have a normal level of RNase III, whether they were isolated at 45 ° C on rich medium plates, or at 37 ° C on plates containing 6-methylaminopurine. In order to find out if the RNase III enzyme in RNase III + revertants behaves like the wild type enzyme, extracts from r n c - parental strains, RNase III+ revertants and r n c + strains were prepared, incubated at various temperatures from 37 to 55°C for 5 minutes, and then assayed at 30 ° C. In Figure 1 the result o f one such experiment is presented. It can be seen that the enzyme from the revertant strain is heat inactivated identically with that of the rnc + strain. Notice that the activity in the r n c - s t r a i n is more heat stable than the activity in rnc + strains. This and other experiments (Apirion et al., 1976; see Discussion) suggest that the activity in extracts of rnc105 strains against double stranded R N A is not due to RNase III. With respect to growth, as was mentioned above, all the R N a s e I I I - T s + revertants grow slower than rnc + strains, while the RNase III+ revertants show normal growth rates. The growth rates of some of the strains were measured at 37 and 45 ° C, and as can be seen in Table 2, the RNase I I I - Ts + revertants grow slower at both temperatures as compared to rnc + strains, but faster as compared to r n c - strains. Previously (Apirion and Watson, 1974, 1975b), we found a significant reduction in the plating efficiency of bacteriophages T4, T7 and )~ on r n c - strains. Therefore, we repeated such experiments with the revertant strains. Again we found some differences between rnc ÷ and r n c - strains, but we noticed that when we used very fresh T4 and T7 stocks the differences were rather small. However, since it was shown that the development of 2 in rnc strains is delayed and the burst size is reduced by a factor of ten (I. Hershkowitz, personal communication; H. Lozeron, personal communication), and since in r n c - strains some of the early 2 R N A transcript becomes stabilized (Lozeron, Anevski, and Apirion, T.M.B. in press), we decided to measure plaque forming ability of 2 on some of the revertants. We tested it in five different
5O
O
O
i--ii ;eve,,oo,
\
2o
Revertants from RNase III Negative Strains of Escherichia coli David Apirion, Jeff Neff, and Ned Watson Washington University Medical School, Department of Microbiology and Immunology, Division of Biology and Biomedical Sciences, St. Louis, Missouri 63110, USA
Summary. E. coli strains carrying the r n c - 1 0 5 allele do not show any level of R N a s e III in extracts, grow slower than rnc + strains at temperatures up to 45 ° C and fail to grow at 45 ° C. Revertants which can grow at 45 ° C were isolated. The vast majority of them still do not grow as fast as rnc + strains and did not regain R N a s e III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of R N a s e III. They do not harbor the r n c - 1 0 5 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate r n c + p u r revertants from an r n c - p u r - strain with relative ease. They behaved exactly like the true rnc ÷ revertants isolated from r n c - strains at 45 ° C. A merodiploid strain which contains the rnc + gene on an episome behaves exactly like an rnc + strain with respect to growth and R N A metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc + strain; thus the level of R N a s e III is not limiting in the cell. The m c - strains show a characteristic pattern of transitory molecules, related to r R N A , 30S, 25S, " p 2 3 " and 18S, which are not observed in rnc ÷ strains. This pattern is unchanged in r n c - strains and in the revertants which are still lacking RNase III, regardless of the temperature in which R N A synthesis was examined (30 ° to 45 ° C). On the other hand, in the mc + strains as well as in the true revertants and the r n c + / r n c - merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase I I I - strains are due to pleiotropic effects of the r n c - 1 0 5 allele, and that the enzyme R N a s e I I I is not essential for the viability of the E. coli cell.
Introduction E. coli strains which carry the r n c - 1 0 5 allele do not
show a significant level of R N a s e III activity in cell
extracts (Kindler, Keil and Hofschneider, 1973; Apirion and Watson, 1974, 1975a, b; Apirion, Neil and Watson, 1976). The original strain AB30 I- 105 isolated by Kindler et al., besides being RNase I I I - , contains a large number of mutations (Apirion and Watson, 1974, 1975b). Therefore when it was shown that strain AB301-105 has an abnormal r R N A metabolism (Dunn and Studier, 1973; Nikolaev et al., 1973) it became necessary to establish to what extent the mutation affecting RNase III is also altering r R N A synthesis. While previous studies (Apirion, 1974; Apirion, Gegenheimer and Watson, 1975; Apirion, Nell and Watson, 1976) indicated that the r n c - 1 0 5 mutation is causing all these pleiotropic effects, they could not rule out the possibility that another mutation very closely linked to r n c - 1 0 5 might be involved in some of these effects. Such a possibility could have been entertained with some credence since previous studies showed that the original r n c - 1 0 5 strains carried a second mutation which did not affect the level of RNase III, but conferred inability to grow at 4 5 ° C (Apirion and Watson, 1975 b) and affected R N A metabolism, probably the maturation of 17S to 16S r R N A (Gegenheimer and Apirion, unpublished observations). In the studies reported here we describe the isolation of revertants and their properties. We show that indeed the r n c - 1 0 5 mutation is causing all the pleiotropic effects found in R N a s e I I I - strains. These studies also suggest that strains carrying the r n c - 1 0 5 allele do not contain RNase III and therefore this enzyme is not essential for the viability of the E. coli cell.
Material and Methods Strains. All strains used are described in Table 1. Media were described previously (Apirion, 1966; Lenncne and
Apirion, 197i), except for low phosphate rich medium which was prepared as follows: Eight grams of nutrient broth and 8 grams of casamino acids were dissolved in 800 ml of water and the pH was adjusted to 10.5 with concentrated NH4OH. Sixty six ml of 1M MgC12 was added dropwise with stirring over a period of 15 minutes. The mixture was then allowed to stir for an additional 30 minutes. The precipitate formed was collected on a Whatman
202
D. Apirion et al. : Revertants from RNase III- Strains
Table 1. Strains used
Strain
Markers
Source
PA3306
F - t h i - i pur I66 argH1 nadB4 lac Y1 g a l - 6 m a l A 1
Lavalle"
N2076 N2077 N2078 N2080 N2082 N2084 N2087 N2088 N2089 N2090 N2097 N2100 N2403 N2416 N2419 N3705
a b c d
2R x y l - 7 a r a - t 3 m t l - 2 str-9 t o n A 2 ? 2 supE44 R F See PA3306 a b o v e , pur + ranA + rnc + F - See PA3306 above, p u t + rne-105 ranA + F - See PA3306 above, pur + nad+ rnc-105 ranA + F rne-105 spontaneous revertant F - rne-105 spontaneous revertant F See PA3306 above, pur + n a d + rne-I05 ranA + F - rne-105 spontaneous revertant
F - rne + spontaneous revertant F - lae + derived from strain N2076 F - lae + derived from strain N2077 F - mal + a spontaneous derivative F See PA3306 above, p u r l nad+ rnc-I05 ranA + F - rnc-105 spontaneous revertant F - rnc + spontaneous revertant F - rnc + spontaneous revertant F' chromosome like in strain N2100 episome F' 142 tyr s u p N (Low, 1972)
N2066~PA3306 b c N2066--+PA3306 N2066~PA3306 from strain N2077 d from strain N2078 d N2066~PA3306 from strain N2084 d from strain N2084 d D10~N2076 d D10--,N2077 a from strain N2090 a N2084~PA3306 d from strain N2077d from strain N2077 d from strain N2100 a KLF42/KL253 x N2100 d
The n o n N strains were supplied by B.J. Bachmann, Yale University, Coli Genetic Stock Center Denotes a transduction; arrow leads from donor to recipient Apirion and Watson, 1975b This paper
No. 5 filter paper. The filtrate was autoclaved for 3 minutes. The resulting solution was filtered again on W h a t m a n No. 5 filter paper, the pH was adjusted to 7.0 with concentrated HC1, the final volume was adjusted to 1.0 liter, and the medium was autoclaved. Protein Determination. This was carried out according to the procedure of Ehresmann, Imbualt and Weil (1973). All other techniques used were previously described. For P1 mediated transduction see, Apirion and Watson (1975), Lennette and Apirion (1971). For assaying RNase III see Apirion and Watson (1974) Apirion et al. (1976). For labeling cells with 32Pi, preparation of extracts, and electrophoresis in polyacrylamide gels, see Gegenheimer and Apirion (1975), Kaplan and Apirion (1975) and Meyhack et al. (1973).
Results I s o l a t i o n o f Ts + R e v e r t a n t s . a) B y G r o w t h a t 4 5 ° C. We reported earlier (Apirion and Watson, 1974; Apirion and Watson, 1975b) that strains which carry the r n c - 1 0 5 allele cannot grow at 45 ° C. Therefore we isolated Ts + revertants by plating r n c - 1 0 5 cells on rich medium plates and incubating them at 45 ° C. Numerous colonies appeared which regained the capacity to grow at 45 ° C. On purifying and retesting these colonies, they behaved as revertants, since after numerous passages (at least 5) at 37 ° C they did not alter their capacity to grow at 45 ° C. Ts + revertants were isolated from a number of strains carrying the r n c - 1 0 5 allele. All such strains revert with a similar frequency, around 10 -5 to 10 6). In order to measure the reversion rate a number of experiments were carried out, one
of which is described below. Fifty cultures were started from different clones of strain N2077, and about 105 cells from each culture were plated on a rich medium plate at 45 ° C. F r o m the 50 samples, 14 had not Ts ÷ revertants. The reversion rate calculated from the zero class (Luria and Delbruck, 1943) was 3 , 4 x 1 0 -6, whereas the reversion frequency was 3 x 10-6 The revertants did not grow as well as rnc ÷ strains at all tested temperatures. This was first observed in replication tests. To make the distinction easier, colonies were streaked out at 45 ° C on rich medium plates. Such tests showed rather clearly that the single colonies from revertant cells, observed in a streak, were smaller in size than colonies from rnc ÷ strains. (A number of rnc ÷ strains were tested, including N2076, the rnc ÷ isogenic partner of the r n c - strain N2077.) Measuring the doubling times of the revertants showed that they do grow somewhat faster than the r n c - parental strains but slower than rnc + strains (Table 2). Further analysis (see below) showed that these revertants lack RNase III. In order to find out if r n c - 1 0 5 is likely to be a point mutation, we tried to isolate true revertants. This was accomplished by plating large numbers of r n c - cells from independent clones on rich medium plates at 45 ° C and picking out from each clone four large revertants. After retesting these large revertants by replication and streak tests, the more promising candidates were assayed for R N a s e III, and some clearly regain RNase III activity (see below). We tried
D. Apirion et al. : Revertants from RNase I I I - Strains
203
Table 2. Doubling times of various strains in rich medium at different temperatures (min)
Table 3. A reconstitution experiment
RNase III
37 C
45 C
rnc-105
No. of colonies observed
No. of colonies tested
Strain N2076 N2089 N2077 N2084 N2100 N2080 a N2087 a N2403" N2088" N2416 a N2419 a N3705 (rnc+/rnc-)
+ + -
45 46 71 73 77 54 63 61 48 47 49 47
56 61 oo oo co 71 75 77 52 53 52 58
cells plated
Large
Large
No. of
+ + +
+
Revertant strains
to isolate such revertants from three different rncstrains, and succeeded in doing so with all of them (for further analysis of these Ts + revertants, see below). b) By Growth on Ademlne Analogues. In order to elimi-
nate the numerous revertants which did not regain RNase III, we resorted to adenine analogues. We found that a p u r - r n c - cell is disadvantaged when adenine is replaced with limited concentrations of other purine bases. We tested a number of those by auxanographic techniques (Pontecorvo, 1949) and found that a medium containing 2 gg/ml of either xanthine or 6-methylaminopurine (MAP) as a substitute for adenine will allow rnc+purl - cells to grow better than r n c - p u r I - cells. In order to ensure that we would be able to use this technique, we ran reconstitution experiments plating about 80 cells per plate of the p u r I - r n c + strain PA3306 with increasing numbers of the p u r I - r n c strain N2100, from 0 to 107 cells per plate (Table 3). We found that by using xanthine it was possible to recover at least 75% of the PA3306 cells when cells of the N2100 strain did not exceed 105 cells per plate (Table 3). When the N2100 cells were plated 106 per plate, about 50% recovery of PA3306 cells was obtained, but no rnc + cells were recovered when the number of N2100 cells were increased to 107 per plate. The background cells, N2100, were observed as smaller colonies at all concentrations tested, becoming relatively smaller with the increase in cell concentration. This was verified by picking out from the test plates large and small colonies, purifying them and testing them. As excepted, all the originally small colonies were Ys- Nic + (as is N2100) and all the large colonies were Ts + Nic- (as is PA3306). Moreover, five colonies from each of these groups were assayed for RNase III and
Small
Small
FHC +
I'~lC-
r~c +
r~c-
0 103 104 l0 s 106 107
83 78 84 68 32 0
0 320 1700 a a a
10 i0 10 8 l0
0 0 0 2 8
0 0 0 10 0 10 0 8 0 8 Not tested
0 103 104 l05 l06 l07
82 98 97 73 18 10
0 2300 ~ 3000 ~6000 a a
10 8 6 7 15 10
0 2 4 3 0 0
0 0 0 10 0 8 0 10 Not tested Not tested
A
A background of limited growth forming a thin lawn of cells 0.1 ml of a culture of strain PA3306 (rnc+purI66 nadB4) containing about ninety cells, and 0.1 ml of a culture from strain N2100 (rnc-105 purI66 had+) containing various amounts of cells as indicated, were plated on minimal medium plates containing thiamine nicotinic acid and arginine. To one set of plates xantine (A) and to another MAP (B) were added (2 gg/ml). The first set of plates were incubated for four days and the second for six days. A number of large and small colonies were picked up, purified and characterized. Colonies whichgrew slower and were Nad + and Ts were classified as N2100 (rnc-) while colonies which were fast growers, N a d - and Ts + were classified as PA3306 (rnc+)
again all the Ts- colonies were RNase I I I - , while all the Ts + colonies were RNase III +. When MAP was used in a reconstitution experiment, we found it necessary to incubate the plates somewhat longer, 6 rather than 4 days, and that at lower to medium concentrations of rnc- cells (103-105) the distinction between the rnc- and rnc ÷ cells is not clear cut. However, at high concentrations of rnc- cells, 106 and 107 cells per plate, the recovery was only around 20 and 15% respectively (Table 3), but all the distinctly large colonies tested as PA3306 cells (tested as above). To isolate RNase III+ revertants, we plated strain N2100 on plates containing 2 gg/ml of either xanthine or MAP (no adenine) and plates were incubated at 37 ° C. Colonies appeared on both types of plates. Twenty colonies were tested in some detail, 6 which appeared on the xanthine plates and 14 which appeared on the MAP plates. All the six colonies isolated from the xanthine plates resulted from reversion to Pur + and remained T s - . F r o m the 14 isolated from the MAP plates only 1 was a Pur+Ts - revertant, 4 were P u r - T s ÷ and the other nine remained P u r - T s - but became mucoid (which helped them to establish distinct colonies on the background of the non-mucoid
204
D. Apirion et al. : Revertants from RNase III- Strains
cells). In this particular experiment, the reversion frequency to Ts + was 2 x 10- 6. These four strains contain normal levels of R N a s e III (see below).
100
C h a r a c t e r i z a t i o n o f R e v e r t a n t S t r a i n s . A number of revertant strains were chracterized for growth, for the ability to be used as host for various phages, and for levels of the RNase III enzyme. With respect to the level of enzyme, we found invariably that all the slow growing revertants remain RNase I I I - exactly as their parental strains. (We tested during the past two years about 280 independently isolated revertants.) On the other hand, all the normally growing revertants have a normal level of RNase III, whether they were isolated at 45 ° C on rich medium plates, or at 37 ° C on plates containing 6-methylaminopurine. In order to find out if the RNase III enzyme in RNase III + revertants behaves like the wild type enzyme, extracts from r n c - parental strains, RNase III+ revertants and r n c + strains were prepared, incubated at various temperatures from 37 to 55°C for 5 minutes, and then assayed at 30 ° C. In Figure 1 the result o f one such experiment is presented. It can be seen that the enzyme from the revertant strain is heat inactivated identically with that of the rnc + strain. Notice that the activity in the r n c - s t r a i n is more heat stable than the activity in rnc + strains. This and other experiments (Apirion et al., 1976; see Discussion) suggest that the activity in extracts of rnc105 strains against double stranded R N A is not due to RNase III. With respect to growth, as was mentioned above, all the R N a s e I I I - T s + revertants grow slower than rnc + strains, while the RNase III+ revertants show normal growth rates. The growth rates of some of the strains were measured at 37 and 45 ° C, and as can be seen in Table 2, the RNase I I I - Ts + revertants grow slower at both temperatures as compared to rnc + strains, but faster as compared to r n c - strains. Previously (Apirion and Watson, 1974, 1975b), we found a significant reduction in the plating efficiency of bacteriophages T4, T7 and )~ on r n c - strains. Therefore, we repeated such experiments with the revertant strains. Again we found some differences between rnc ÷ and r n c - strains, but we noticed that when we used very fresh T4 and T7 stocks the differences were rather small. However, since it was shown that the development of 2 in rnc strains is delayed and the burst size is reduced by a factor of ten (I. Hershkowitz, personal communication; H. Lozeron, personal communication), and since in r n c - strains some of the early 2 R N A transcript becomes stabilized (Lozeron, Anevski, and Apirion, T.M.B. in press), we decided to measure plaque forming ability of 2 on some of the revertants. We tested it in five different
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