May 21, 1973 - 'Present address: Department of Microbiology, Uni- versity of Massachusetts Medical School, Worcester,. Mass. 01604. ts-14, can grow ... NOTES. 400. 300. 100. 50 so. 10 so0. 10 -. 10. 20. D 100. 90 e 70. Y. 60. 50. 40. 30. 20.
JOURNAL OF BACTERIOLOGY, Aug. 1973, p. 703-706 Copyright © 1973 American Society for Microbiology
Vol. 115, No. 2 Printed in U.S.A.
NOTES
Ultrastructural Studies of Sporulation in a Conditionally Temperature-Sensitive Ribonucleic Acid Polymerase Mutant of Bacillus subtilis LEATRICE SANTO, TERRANCE J. LEIGHTON,1
AND
ROY H. DOI
Department of Biochemistry and Biophysics, University of California, Davis, California 95616
Received for publication 21 May 1973
Morphological studies of a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis have revealed that sporulation is inhibited at stage II when the cells are grown at 47.5 C. Growth and sporulation occur normally at 30 C with the mutant. The mutant grows normally at 47.5 C but is prevented from sporulating at the nonpermissive temperature by an abnormal septation during forespore membrane formation which prevents the subsequent engulfment process (stage III). The mutation affects the normal functioning of ribonucleic acid polymerase at the nonpermissive temperature resulting in abortive sporulation. ts-14, can grow and sporulate normally at 30 C; at 47.5 C it can grow at the same rate as the wild type, but cannot sporulate. Its RNA synthesis pattern is identical to the wild-type pattern at 30 C, but differs dramatically at 47.5 C during sporulation time periods. Preliminary investigation by light microscopy suggested that these mutants could form a forespore at 47.5 C. This fine structure analysis was undertaken to determine at which step sporulation was inhibited in this conditional RNA polymerase mutant. B. subtilis W168 and B. subtilis W168 ts-14, a rifampin-resistant temperature-sensitive sporulation mutant (2), were grown and fixed as previously described (7). At the permissive temperature (35 C), both strains W168 and ts-14 had similar growth rates and spore yields (Fig. la), but the onset of sporulation in ts-14 was delayed 1.5 h (Fig. 2). The ultrastructural events occurring during sporulation of W168 and ts-14 appeared to be similar (or identical) to the process previously described for B. subtilis WB746 (7). At the non'Present address: Department of Microbiology, Uni- permissive temperature (47.5 C), the wild type versity of Massachusetts Medical School, Worcester, sporulated normally. The ts-14 cells had a similar growth rate as the wild type during Mass. 01604.
Biochemical and genetic investigations have established that Bacillus subtilis ribonucleic acid (RNA) polymerase molecular structure and template specificity are modified early in sporulation (1, 4-8). This modification is apparently effected by a serine protease which is derepressed at the end of the log phase of growth (4; Leighton et al., J. Mol. Biol., in press, 1973). Ultrastructural examination of a temperaturesensitive serine protease mutant indicates that these cells do not progress beyond stage 0 or I at the nonpermissive temperature (7). From these studies, it is clear that RNA polymerase modification is required prior to forespore membrane formation. Since a sequence of biochemical and morphological events occurs during sporulation, it appeared that other biochemical modifications which affect RNA polymerase structure or function were possible. To test for such a possibility, an RNA polymerase mutant which is conditionally temperature sensitive only during sporulation has been isolated and characterized by Leighton (2). This mutant, B. subtilis W168
703
J. BACTERIOL.
NOTES
704
I
I
--I
+
6,00
la
I
I.
I
lb
400
00
I~ ~ ~ ~ ~ ~
300 00
/F
0*
tI
/
~00 D 100 90 :
100
11
50
e 70
100 90 so
70
so. Y,
Y
10
I
60
60
/.
50
so0
40
//
40
10
0~~~~, 10 so
-
30
30 _
20
20
10
300
20
20
10_
10
i 0
2
4
6
a T
10
12
m e
(H
14
16
18
20
.do
s§)
-0 u
2
I --A- t -I 4 6 a
e
-
I
E
10
12
T, meI
(H
14 I
16
1
20
22
s)
FIG. 1. Growth and sporulation curves for strains W168 and ts-14. (a) Growth at 35 C. (b) Growth at 47.5 C. The closed and open circles represent strains W168 and W168 ts-14, respectively. The arrows indicate the sample times for the electron micrographs presented in this paper.
exponential growth (Fig. lb), but the normal sporulation sequence was not observed at 47.5 C. When wild-type cells were at stage III (forespore engulfmefit stage), we observed only asymmetric septum formation in ts-14 (Fig. 3). Cell wall material which is not seen in normal sporulation was also present in this septum. Engulfment may have been attempted (Fig. 4), but forespores were never observed. Prolonged incubation resulted in the lysis of both protoplasts, leaving "ghosts" or cell wall skeletons of the aborted forespore and the mother cell (Fig. 5). Normal sporulation beyond this stage was never observed with ts-14 at the nonpermissive temperature.
The cytological data suggest that sporulation is affected during stage II when the mutant ts-14 is grown at 47.5 C. The forespore membrane and the nascent forespore protoplast are formed at this temperature. However, an abnormal synthesis of cell wall material occurs along the forespore membrane which precludes any interaction between the nascent forespore protoplast and the mother cell protoplast, and prevents the normal engulfment process (Fig. 4). Since engulfment cannot occur, the sporulation process is inhibited, and the walled-off nascent forespore protoplast eventually lyses
(Fig. 5). Although these results can also be interpreted as simply an abnormal septation phenomenon, the time of formation and the size of the newly enclosed protoplast are clearly correlated with the stage of forespore membrane and protoplast formation at stage II. The data of Leighton (2) indicate that the temperaturesensitive nature of the mutant is due to a mutation in RNA polymerase. These observations suggest, therefore, that the nonpermissive temperature affects the correct functioning of the mutated RNA polymerase during or before stage II of sporulation. Several explanations are possible for the malfunctioning of the temperature-sensitive RNA polymerase which would result in the abnormal production of cell wall material during forespore membrane formation. (i) The mutant RNA polymerase cannot transcribe a gene required for the regulation of cell wall synthesis at the end of the growth phase. (ii) The mutant RNA polymerase has an enhanced specificity for the promoter of the structural and/or regulatory genes involved in cell wall synthesis or structure. (iii) The mutant RNA polymerase cannot be modified correctly to alter its transcriptional specificity for stage III sporulation genes and subsequently directs abnormal synthesis of var-
( 4
FIG. 2-5. Growth of strain ts-14 at the nonpermissive temperature (47.5 C). The bar indicates 0.5 ,um. (Fig. 2.) A vegetative cell at the time when the W168 shows forespore suptum formation. A mesosome (M) can be seen. (Fig. 3.) Asymmetric septum formation with deposition of cell wall material. The arrows indicate another septum initiation site. (Fig. 4.) Asymmetric septum formation where engulfment may have been attempted. (Fig. 5.) A ghost after release of cellular material; the cell wall remains. Note the remnants of the septum cell wall material. 705
706
NOTES
ious proteins including any involved in cell wall synthesis. (iv) The mutant RNA polymerase cannot interact with effectors which regulate its transcriptional specificity towards sporulation genes resulting in the expression of some vegetative functions. These possibilities can all arise from an altered conformation of the mutant RNA polymerase at the nonpermissive temperature and a continuing function of cross wall synthesizing enzymes in stages 0 to II (7). There exists an interesting correlation between the continued expression of cross-wallsynthesizing genes and the overproduction of RNA (2) seen in the mutant cells at 47.5 C. It is possible that a class, or classes, of vegetative genes is not correctly turned off in this mutant. Our data also would suggest that the inhibition of cross wall information represents a regulatory event involving RNA polymerase site selection. It is evident that sporulating cells have the capacity, under abnormal circumstances, to progress through an asymmetric cell division complete with cross wall formation. It appears that the inhibition of this occurrence is a stringently regulated process, rather than a result of the changes in secondary metabolism characteristic of postexponential cells. The inhibition of sporulation in conditionally temperature-sensitive RNA polymerase mutants at stage II with ts-14 and at stages I and III (Sumida, Doi and Santo, unpublished observations) suggest that several modifications of RNA polymerase or interactions of RNA polym-
VOL.
115, 1973
erase with effectors may occur during sporulation. The RNA polymerase of the wild type and these mutants are being investigated to determine whether this is indeed the case. This research was supported by the National Science Foundation grant GB-26409 and Public Health Service grant GM-19673-01 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Brevet, J., and A. L. Sonenshein. 1972. Template specificity of ribonucleic acid polymerase in asporogenous mutants of Bacillus subtilis. J. Bacteriol. 112:1270-1274. 2. Leighton, T. J. 1973. An RNA polymerase mutation causing temperature-sensitive sporulation in Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 70:1179-1183. 3. Leighton, T. J., and R. H. Doi. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J. Biol. Chem. 246:3189-3195. 4. Leighton, T. J., P. K. Freese, R. H. Doi, R. A. J. Warren, and R. A. Kelln. 1972. Initiation of sporulation in Bacillus subtilis: requirement for serine protease activity and RNA polymerase modification, p. 238-246. In H. 0. Halvorson, R. S. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washingtpn, D. C. 5. Losick, R., R. G. Shorenstein, and A. L. Sonenshein. 1970. Structural alteration of RNA polymerase during sporulation. Nature (London) 227:910-913. 6. Losick, R., and A. L. Sonenshein. 1969. Change in the template specificity of RNA polymerase during sporulation of Bacillus subtilis. Nature (London) 224:35-37. 7. Santo, L., T. J. Leighton, and R. H. Doi. 1972. Ultrastructural analysis of sporulation in a conditional serine protease mutant of Bacillus subtilis. J. Bacteriol. 111:248-253. 8. Sonenshein, A. L., and R. Losick. 1970. RNA polymerase mutants blocked in sporulation. Nature (London) 227:906-909.
