Feb 6, 1973 - and Simon (12). ..... Hanson, R. S., Peterson, J. A. & Yousten, A. A. (1970). Annu. Rev. ... Hanson, R. H. & Campbell, L. L. (American Society for.
Proc. Nat. Acad. Sci. USA Vol. 70, No. 4, pp. 1179-1183, April 1973
An RNA Polymerase Mutation Causing Temperature-Sensitive Sporulation in Bacillus subtilis (template specificity/rifampin-resistant mutants/altered sporulation/RNA synthesis)
T. J. LEIGHTON University of Massachusetts Medical School, Worcester, Mass. 01604
Communicated by Michael Doudoroff, February 6, 1973 A single-site mutant of Bacillus subtilis ABSTRACT with a rifampin-resistant RNA polymerase has been isolated; this mutation causes temperature-sensitive sporulation. The temperature-sensitive mutation was only expressed during a limited time period, covering the middle third of the sporulation sequence. Mutant cells grown at the nonpermissive temperature exhibited the normal change in RNA polymerase template specificity, accumulated extracellular proteolytic activity and antibiotic activity, but failed to accumulate alkaline phosphatase and, hence, were blocked at or near stage III in the sporulation sequence. Pulse-labeled RNA synthesis was seriously deranged during postexponential growth phase in mutant cells incubated at the nonpermissive temperature.
Bacterial sporulation represents an ideal system in which to study the differential control of gene expression during a simple process of cell differentiation. Bacillus subtilis is particularly suitable for investigations of this type, since a great deal is known about the genetics (1, 2), physiology (3, 4), and biochemistry (3-6) of the developmental process. Previous studies have indicated that some form of transcriptional control must be operative during spore formation, since unique mRNA species are found in sporulating cells (6). Furthermore, it is known that a proteolytic cleavage of the 3 subunit of RNA polymerase is an absolute requirement for sporulation to proceed past state 0 (7-9). Mutants with an altered serine protease (7) or an altered RNA polymerase (8, 9) block sporulation at stage 0 by interfering with the clippage of 3 subunit. An important and unanswered question is whether sporulation-specific alterations in RNA polymerase molecular structure are necessary at other stages in the sporulation process. This communication establishes that a mutation of RNA polymerase can specifically affect development over a limited time during midsporulation.
genized with ethane methyl sulfonate to 80% survival (11). Survivors were plated onto tryptose-blood agar-base plates at 35°. Incubation was continued for 4 hr at 35°. The plates were then spread with 0.2 ml of a 700 ,g/ml solution of rifampin. Incubation was continued until resistant colonies appeared. All colonies were replicated onto similar plates at 350 and 480. Asporogenous mutants lyse on such plates after 24-48 hr of incubation (7). Those strains that formed normalsized colonies at 480, which subsequently cleared, and that formed normal-sized opaque colonies at 350 were characterized further. Spontaneously occurring revertants of the rifr spot" strains were isolated as opaque colonies that appeared after prolonged incubation on plates at 480 (7). Genetic transduction with bacteriophage PBS1 was done as described by Van Alstyne and Simon (12). Donor transducing lysates were prepared from mutant cells and were used to transduce the cys- or gua strains to prototrophy. The number of rifampin-resistant transductants among the prototrophs was used to estimate the cotransduction frequency. Temperature-shift experiments, to determine the time period during which the temperaturesensitive mutation was expressed, were performed as described
(7). RESULTS
Using the described mutagenesis and screening procedure, I have obtained 106 mutants that have the phenotype of being rifampin resistant and temperature sensitive for sporulation. The properties of one of these mutants, ts-14, will be described here in detail. As can be seen from Fig. 1, ts-14 cells grew and sporulated normally at 350, and grew normally but did not sporulate when incubated at 460. About 0.1% of the ts-14 cells present at to (1.5 hr at 460) produced heat-resistant spores when assayed after 24 hr of incubation. About 80% of the wild-type cells present at to (460) produced heatresistant spores in a parallel experiment. Spontaneous sporogenous revertants of ts-14 occurred at a frequency of 1-3 in 106 cells and were sensitive to rifampin (25/25). The ts-14 mutation cotransduced at 80% frequency with the cys marker, and at 15% frequency with the gua marker. These two-factor crosses place the mutation in the rif locus of Bacillus subtilis (2). All cys+, rifr (50/50) or gua+, rift (35/35) transductants isolated were temperature sensitive for sporulation. Hence, the rifr and spot" genotypes were tightly linked. A series of shift-up and shift-down experiments (Fig. 2) es-
MATERIALS AND METHODS
Bacillus. s-sbtilis W168 was grown (7, 10) in a modified Schaeffer's Medium. Bacillus subtilis cys and bacteriophage PBS1 were from L. R.'Brown, and Bacillus subtilis trp-, guawas from J. A. Hoch. Procedures for the estimation of refractile bodies (10), protease accumulation (7), antibiotic activity (7), alkaline phosphatase accumulation (10), RNA synthesis (7), and RNA polymerase template activity (7) were described. Temperature-sensitive sporulation (spot") mutants with defects in RNA polymerase were isolated by screening for a subclass of rifampin-resistant (rifr) mutants with the expected properties. Bacillus subtilis W168 spores were muta-
1179
1180
Microbiology: IAghton
Proc. Nat. Acad. Sci. USA 70 (1978)
40)
c
c
m ID
0
xy
-100
80 .X -60 S -40 .!
I0
-_0
0
D
0
32
-20 I
Ir
a
Hours
FIG. 1. Growth and sporulation of W168 and W168 ts-14. Cells were grown in a modified Schaeffer's Medium containing 0.1% glucose (10). (a) Growth and sporulation of W168 (@) and W168 ts-14 (0) at 35°. (b) Growth and sporulation of W168 (@)'and W168 ts-14 (0) at 46°.
a c ;, Time of temperature shift
I
(Ir)
W168t-S-14 L
0.0
I
I
0.2
0.4
t INITIATION
A
I
0.6
b I
I
I
0.8
1.0
+~~~~~~~~~ TERMINATION
FRACTION OF THE SPORULATION SEQUENCE
FIN 2. Estimation of the ts-14 temperature-sensitive period. (a) Sporulation of t8-14 cells after a shift-down from 460 to 350 (0) or a shift-up (0) from 35° to 46°. Cultures were incubated for 24 hr after the temperature shift, and refractile bodies were estimated (10). (b) Physiological map of the ts-14 temperaturesensitive period. Data from the temperature-shift experiments were normalized to a common time scale, as described by Espositoetal. (19).
tablished that the mutation was only expressed during a limited time period, covering the mid-third of the sporulation sequence. The accumulation of serine and metal proteolytic activities in the culture supernatant fraction, an early sporulation event (1, 7, 13-15), occurred with a similar time course in mutant and wild-type cells at 350 and 460 (Fig. 3). As judged by appropriate inhibitor experiments, at any given time 75% of the azocasein activity was due to serine protease and 25% of the activity was due to to metal protease (14). The mutant cells slightly underproduced both enzymes at 35° and 460. Both mutant and wild-type cells produced antibiotic activity, another early sporulation event (13), at 350 and 460 (data not shown). Alkaline phosphatase accumulation, which begins at stage III (16), was similar in both ts-14 and W168 cells grown at 350 (Fig. 4). However, at 460, ts-14 cells failed to exhibit the sporulation-associated increase in alkaline phosphatase activity. Pulse-labeled RNA profiles were seriously deranged in ts-14 cells grown at 460 (Fig. 5). Pulse-labeled RNA was synthesized similarly to the W168 pattern in ts-14 cells grown at 35°. RNA polymerase from vegetative cells of W168 and ts-14 actively transcribed both bacteriophage Be DNA and poly(dA-dT) in vitro (Table 1). However, sporulation enzyme from either strain grown at 350 or 46° did not transcribe 0e DNA nearly as well as poly(dA-dT). RNA polymerase from ts-cells was rifampin-resistant in vitro. When equal amounts of W168 and W168 ts-14 extracts were mixed and incubated with rifampin, only the part of the activity found in rifampin-inhibited to14 extracts was expressed. This result suggests that the rifampin resistance of ts-14 cells is due to a structural alteration of the RNA polymerase molecule, rather than to an activity in ts-14 cells that inactivates rifampin.
Proc. Nat. A cad. Sci. USA 70
I
RNA Polymerase and Sporulation
(1973) 10080*
E
I
60*
"I..
b
a
p
100' 8060
c
'E
D
40-
:5
6 8 10 12 Hours
2 4
6 8 Hours
2 4
10 12
FIG. 3. Protease accumulation in W168 and W168 ts-14. (a) Proteolytic activity of total culture supernatant fraction directed against azocasein for W168 (0) and W168 ts-14 (0) grown at 35°. (b) Proteolytic activity of total culture supernatant fraction directed against azocasein for W168 (0) and W168 ts-14 (0) grown at 46°. One unit of proteolytic activity hydrolyzes 1 mg of azocasein per hr at 37°. 600
4020-
20-
2 4
2 4
6 8 10 12 Hours
"T
-
200
-
6 8 I0 12 Hours
FIG. 4. Accumulation of alkaline phosphatase in wild-type and mutant cells. (a) Alkaline phosphatase activity in W168 (0) and W168 ts-14 (0) grown at 35°. (b) Alkaline phosphatase activity in W168 (@) and W168 ts-14 (0) grown at 46°. One unit of alkaline phosphatase activity will hydrolyze 1 nmol of pnitrophenylpyrophosphate per min per mg of protein at 37°.
b
aI 400
1181
100
80 -
it)
I0 60 x
-
I0 I I
40-
0.
20 -
I08 6
4-
2
3
4
6 5 Hours
7 8
2
3
4
5 6 Hours
7
8
FIG. 5. Pulse-labeled RNA synthesis in W168 and W168 ts-14. (a) RNA synthesis in W168 (closed bars) and W168 ts-14 (open bars) grown at 35°. (b) RNA synthesis in W168 (closed bars) and W168 ts-14 (open bars) grown at 460. Duplicate 0.5-ml samples of cells were exposed to a 3-min pulse of [5-3H] uridine (25 Ci/mmol), 40 MACi/ml, plus 5 .ug of carrier uridine. Incorporation was stopped by the addition of 10 ml of 5% trichloroacetic acid containing 500 ug/ml of uridine, and samples were processed for scintillation counting (10). Zerotime backgrounds have been subtracted.
1182
Microbiology: Leighton
Proc. Nat. Acad. Sci. USA 70 (1973)
TABLE 1. RNA polymerase activity on exogenous templates Growth temperature
350 Units
460 Units
poly(dA-dT) Source of extract Mid-log W168 Mid-log W168 ts-14 Postexponential W168 Postexponential W168t-14 Postexponential W168 plus postexponential W168 ts-14
qoe 35.1 32.7 5.3 2.9
poly(dA-dT) 15.2 15.0 16.7 16.3
plus rifampin 0.1 15.3 0.1 16.7
30.7
15.9
30.4 28.7 6.1 3.4
poly(dA-dT) 14.9 14.1 16.3 16.1
poly(dA-dT) plus rifampin 0.1 14.5 0.1 16.9
-
32.1
17. o
oe
Samples in the mid-logarithmic phase of growth were taken after 1 hr of growth at 350 or 0.75 hr of growth at 460. Postexponential samples were taken 3.0 hr after to (to = 2.0 hr of growth at 350 or 1.5 hr of growth at 460). One unit of RNA polymerase activity incorporates 1 nmol of [3H]UMP per hr at 37°. Rifampin was present, where applicable, at 0.5 /g/ml.
DISCUSSION The ts-14 phenotype is due to a single-site mutation in RNA polymerase that specifically affects the sporulation process. When ts-14 cells are grown at the nonpermissive temperature, early sporulation functions appear in the normal sequence. However, the sporulation-associated increase in alkaline phosphatase activity (a stage III event) does not occur in these cells. Furthermore, the typical increase in pulse-labeled RNA synthesis, occurring in wild-type cells after stage III, is not seen in ts-14 cells grown at 460. Hence, these cells appear to be blocked biochemically and physiologically at or near stage III. The period of temperature sensitivity is consistent with these data, as is electron microscopic evidence (Santo, Leighton, and Doi, manuscript in preparation). It is interesting to compare and contrast this mutant phenotype to that of a temperature-sensitive mutant in serine protease (ts-5) that blocks sporulation at stage 0 (7). Growth at the nonpermissive condition, for both mutant types, results in a failure to accumulate alkaline phosphatase and heat-resistant spores. However, in ts-5 cells, serine protease activity is underproduced, RNA polymerase is not modified due to a temperature-sensitive alteration in the serine protease, Be DNA is actively transcribed by postexponential extracts, and antibiotic activity is not produced. In contrast, ts-14 cells produce all proteolytic enzymes and antibiotic activity, and they cease to transcribe 4e DNA during postexponential time periods. However, the initial derangement in postexponential pulse-labeled profiles of RNA are similar in both mutants. RNA synthesis does not decrease in the wild-type manner during postexponential phase, nor does it continuously rise again after stage III. The amount of [8H]uridine found in the intracellular pool is similar in both mutant and wild-type strains (unpublished data); hence, differential abilities to concentrate exogenous uridine are not an explanation for these results. I have not measured the specific activity of the UTP pool in any of these experiments. Hence, the variations in incorporation do not necessarily reflect changes in the rate of RNA synthesis. Furthermore, this type of experiment cannot differentiate classes of RNA being synthesized. The utility of these experiments are that they provide a physiological assay for sporulation-associated changes in RNA synthesis. The patterns are reproducible in
wild-type cells and are deranged at the expected time period in all mutant cells I have examined. The RNA synthesis profiles are qualitatively similar in wild-type cells grown at 350 or 46°, when one takes into account the fact that cells growing at 460 start sporulating 0.5 hr sooner than cells growing at 350. However, cells grown at 460 consistently demonstrate more dramatic quantitative variations in incorporation during sporulation time periods than do cells grown at 35°. Possibly, both ts-5 and ts-14 cells fail to turn off certain vegetative genes that are usually not transcribed during sporulation time periods, such as ribosomal genes (17, 18). It has been demonstrated that a rifampin-resistant mutation of RNA polymerase (rfr-10) that blocks sporulation at stage 0 causes continued transcription of 4e DNA and ribosomal genes (8, 17, 18) during postexponential time periods. Wild-type sporulating cells do not transcribe Oe DNA or ribosomal genes in vivo, and RNA polymerase prepared from these cells exhibits similar specificity in vitro (8, 17, 18). It may be that other classes of vegetative genes are turned off by additional mechanisms and that the ts-14 mutation interferes with these processes. The ts-14 RNA synthesis profile differs from the ts-5 profile at midsporulation time periods, as there is a significant increase and subsequent decrease in incorporation not seen in ts-5 cells. This pattern may be related to the later block in the sporulation process seen in these cells. Although the RNA polymerase modification that is blocked in ts-5 seems to occur in ts-14, these cells are still not able to sporulate. This finding suggests that additional control of development must be exerted by RNA polymerase at stage III. To resolve whether the ts-14 phenotype is due to a failure to interact with sporulation-specific factors similar to sigma, a failure to be structurally modified in some unknown manner, or simply that after the modification of , subunit the polymerase fails to transcribe certain types of sporulation genes as a direct result of mutation, will require much more detailed studies of the molecular structure of RNA polymerase at these time periods. The ts-14 mutant is only one of a large number of mutants that are rifampin resistant and temperature sensitive for sporulation. In preliminary experiments, many of these mutants appear to block sporulation at uniquely different
Proc. Nat. Acad. Sci. USA 70
RNA Polymerase and Sporulation
(1973)
time periods including stage 0, stage II, stage IV, etc. Some of the mutants have long temperature-sensitive periods and others have very short, discrete temperature-sensitive periods. Hence, it would appear that RNA polymerase is specifically involved in the regulation of sporulation at many different stages. Hopefully, this collection of mutants will offer a means to study how cellular development is controlled by RNA polymerase in this simple organism. I am grateful to R. H. Doi and D. J. Tipper for the use of their facilities during parts of this investigation. I thank Richard Losick for his critical reading of the manuscript, and acknowledge the expert technical assistance of Dwight Simpson. This research was supported by a General Research Support Grant (RR 0571201) from the University of Massachusetts Medical School. 1. Schaeffer, P. (1969) Bacteriol. Rev. 33, 48-71.. 2. *Young, F. E. & Wilson, G. A. (1972) in Spores V, eds. Halvorson, H. O., Hanson, R. H. & Campbell, L. L. (American Society for Microbiology, Washington, D.C.), pp. 77106. 3. Hanson, R. S., Peterson, J. A. & Yousten, A. A. (1970) Annu. Rev. Biochem. 24, 53-90. 4. Dawes, I. W. & Hansen, J. N. (1972) Critical Rev. Microbiol. 1, 479-520. 5. Kornberg, A., Spudich, J. A., Nelson, D. L. & Deutscher, M. P. (1968) Annu. Rev. Biochem. 37, 51-78. 6. Doi, R. H. & Leighton, T. J. (1972) in Spores V, eds. Halvorson, H. O., Hanson, R. H. & Campbell, L. L. (Amer-
7.
8. 9. 10. 11.
12. 13. 14. 15.
1183
ican Society for Microbiology, Washington, D.C.), pp. 225-232. Leighton, T. J., Freese, P. K., Doi, R. H., Warren, R. A. J. & Kelln, R. A. (1972) in Spores V, eds. Halvorson, H. O., Hanson, R. H. & Campbell, L. L. (American Society for Microbiology, Washington, D.C.), pp. 238-246. Sonenshein, A. L. & Losick, R. (1970) Nature 227, 906-909. Losick, R., Shorenstein, R. G. & Sonenshein, A. L. (1970) Nature 227, 910-913. Leighton, T. J. & Doi, R. H. (1971) J. Biol. Chem. 246, 3189-3195. Mandelstam, J. & Waites, W. M. (1968) Biochem. J. 109, 793-801. Van Alstyne, D. & Simon, M. I. (1971) J. Bacteriol. 108, 1366-1379. Hoch, J. A. & Spizizen, J. (1969) in Spores IV, ed. Campbell, L. L. (American Society for Microbiology, Bethesda, Md.), pp. 112-120. Millet, J. (1970) J. Appl. Bacteriol. 33, 207-219. Michel, J. F. & Millet, J. (1970) J. Appl. Bacteriol. 33,
220-227.
16. Waites, W. M., Kay, D., Dawes, I. W., Wood, D. A., Warren, S. C. & Mandelstam, J. (1970) Biochem. J. 118, 667-676. 17. Hussey, C., Losick, R. & Sonenshein, A. L. (1971) J. Mol. Biol. 57, 59-70. 18. Hussey, C., Pero, J., Shorenstein, R. G. & Losick, R. (1972) Proc. Nat. Acad. Sci. USA 69, 407-411. 19. Esposito, M. S., Esposito, R. E., Arnaud, M. & Halvorson, H. 0. (1970) J. Bacteriol. 104, 202-210.
