Vol. 125, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 74-83 Copyright 0 1976 American Society for Microbiology
Evolution of the Transcription Complex During Sporulation of Bacillus subtilis JEAN BREVET' Institut de Microbiologie, Universite Paris-Sud, Centre d'Orsay, 91405 Orsay, France Received for publication 29 July 1975
Ribonucleic acid polymerase activity in partially purified extract of cells of Bacillus subtilis harvested at different times (t 1, to, t,, and t2) was studied by zone centrifugation. During the course of sporulation, vegetative sigma-factor activity decreased and the transcription complex lost some of its affinity for active sigma factor. The complex underwent a two-stage change in sedimentation value, from 14.5S in vegetative growth phase to a 13S species very early in sporulation to a 16S species at later times. Two Spo0 mutants have been studied by zone centrifugation. One strain, a rifampin-resistant (RfmR) mutant, failed to show any modification of the transcription complex, whereas the other, a Rfms strain, underwent a partial evolution of the transcription complex after to.
The change in template specificity of ribonu- a t2 extract and separated from the holoenzyme cleic acid (RNA) polymerase, which takes place by diethylaminoethyl-cellulose chromatograduring the first 2 h of sporulation (t0-t2) (10), is phy. correlated with loss of vegetative sigma-factor Through the use of zone centrifugation techactivity (2, 9). Since highly purified core en- niques, it has been possible to demonstrate zyme extracted from cells at t, in the presence physical and functional heterogeneity of RNA of protease inhibitors has the same subunit polymerase in crude extracts of growing cells of composition as vegetative core enzyme (2, 9, 15) Bacillus subtilis (16) and Escherichia coli (23). and responds to the addition of vegetative In the present work, I have looked for possible sigma factor, sporulation core seems to be changes in the transcription system during indistinguishable from vegetative core enzyme. sporulation using zone centrifugation analyses These facts and the recent results of Tjian and of partially purified extracts of cells harvested Losick (22) suggest that interference with bind- at t-l, to, tl, and t2. I show here that during ing or activity of the vegetative sigma factor sporulation there is a decrease in the affinity of rather than structural modification of vegeta- RNA polymerase for the vegetative sigma factor tive core enzyme subunits is responsible for the and that there is a changing pattern in the sedimentation coefficient of the transcription change in template specificity. Many single-site mutations conferring resist- complex. ance to rifamycin derivatives or streptolydigin to the core enzyme affect at the same time the MATERIALS AND METHODS ability to sporulate and the change in specificity Bacterial strains. Spo+ strains used were 168 of the polymerase. The favored interpretation is (trpC2) and M021 (RfmR) (2), and Spo- strains were that transcription of sporulation genes requires SpoOb 6Z (6, 12) and M032 (RfmR, Spoo) (19). changes in the enzyme which may no longer be Bacterial cultures. Bacteria were cultivated at possible when this enzyme is altered by muta- 37 C with vigorous rotary agitation in 2-liter Fernbach tion. Such changes may be alterations in the flasks containing 500 ml of nutrient broth medium enzyme structure or binding of specific factors described by Schaeffer et al. (17). Growth was followed of transcription. These putative factors might turbidometrically at 570 nm with a Zeiss spectrophotometer. Under these conditions, at t. (8 h after the be lost during the course of purification of RNA end of exponential growth) 80% of the cells present at polymerase. One such case was previously de- the beginning of the stationary phase were resistant to scribed (13): a protein fraction that stimulated heating for 10 min at 80 C. t,, of sporulation refers to n transcription of coliphage T7 deoxyribonucleic hours after the end of exponential growth. acid (DNA) preferentially was demonstrated in Buffers. The buffers used are derived from those described by Burgess (4). Buffer G contained 0.05
I Present address: Division of Clinical Pharmacology, Department of Medicine, Stanford University Medical School, Stanford, Calif. 94305.
M
Tris(hydroxymethyl)aminomethane-hydrochloride,
pH 7.9; 0.01 M MgCl,; 0.2 M KCl; 0.1 mM 74
VOL. 125, 1976
ethylenediaminetetraacetate; 0.1 mM dithiothreitol; 10% (vol/vol) glycerol. Buffer A contained 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.9; 0.1 M KCl; 0.1 mM ethylenediaminetetraacetate; 0.1 mM dithiothreitol; stated glycerol concentration. Preparation of celi extracts. One liter of a vegetative culture or 500 ml of stationary-phase culture was centrifuged (15,000 x g for 10 min at +4 C), and the cells (ca. 1 g wet weight) were washed once in buffer G, quickly frozen, and stored at - 70 C until used. Pellets were suspended in 10 ml of buffer G, to which inhibitors of B. subtilis proteases had been added: phenylmethylsulfonyl fluoride (0.5 ml of an alcoholic solution at 12 mg/ml) and ethylenediaminetetraacetate-Mg-Na, (5 mM) (protease inhibitors prepared just before use). The pH was adjusted by the addition of 1 N KOH. The cells were broken by two 2-min exposures to sonication (MSE desintegrator), keeping temperature below 4 C. Ribosomes were removed by centrifugation for 90 min at 100,000 x g. Proteins in the supernatant fluid were precipitated by the addition of 2.1 g of ammonium sulfate per 10 ml, and the precipitate was removed by centrifugation at 100,000 x g for 30 min. Then, 2.1 g of ammonium sulfate per 10 ml was added to the supernatant fluid to give a 60% saturated solution. The precipitate was centrifuged at 100,000 x g for 30 min, and the pellet (SI extract) was dissolved in 1 to 2 ml of buffer A without glycerol. A fraction of this extract was withdrawn for zone centrifugation analysis, and to the remainder an equal volume of buffer A containing 90% glycerol (vol/vol) was added and the extract was stored at - 20 C. Zone sedimentation analysis. One to 2 mg of protein from an SI extract in buffer A without glycerol was immediately applied to a 12-ml linear gradient of 10 to 30% (vol/vol) glycerol in buffer A and centrifuged for 15 h at 38,000 rpm in a Spinco SW41 rotor. Fractions were collected, and 50 Al was assayed for sigma activity or RNA polymerase (Fig. 1, legend). Polymerase assay. The RNA polymerase assay has been described previously (3), with the following modifications: assays using $e DNA as template were done in the presence of 10 mM MgCl,, whereas assays using poly d(A-T) were done in the presence of 1 mM MnCl, (2). ['H ]adenosine 5'-triphosphate was used at 6,000 counts/min per nmol. Sigma activity. The assay conditions for sigma activity were identical to those described above for RNA polymerase activity with $e DNA as template, except that 2 ;&g of pure vegetative core enzyme extracted from strain M021 (RfmR) and rifamycin (1 /ug/ml, a concentration inhibitory to the wild-type enzyme) were added (2). The presence of rifamycin eliminated the contribution of the RNA polymerase activity present in the fractions of the gradient. SDS-polyacrylamide gel electrophoresis. Conditions for sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis were according to Laemmli (8), the polyacrylamide gels (12 cm) being prepared according to Linn et al. (9). Expression of the results. One unit of RNA polymerase activity is defined as the amount of
EVOLUTION OF RNA POLYMERASE
75
enzyme that incorporates 1 nmol of ['H Jadenosine 5'-monophosphate in 10 min at 37 C. One unit of net stimulatory sigma activit& increased the activity of excess vegetative core polymerase with te DNA by one RNA polymerase unit. In analyzing glycerol gradients, values for RNA polymerase activity in the presence of poly d(A-T), RNA polymerase activity in the presence of te DNA, and free vegetative sigma activity were the sums of the activities found in the appropriate fractions of the gradients. From these values several ratios were calculated. R:Mn is the ratio of transcription on poly d(A-T) to transcription on te DNA (2, 3) and is a measure of the template specificity of the enzyme since *e DNA serves as template for only RNA polymerase holoenzyme, whereas poly d(A-T) is transcribed by both holoenzyme and core enzyme. Previous experiments indicated that the activities of vegetative holoenzyme on these substrates produced a R:Mn ratio of 2 to 3 (unpublished data; 2). The ratio free a activity/ d(A-T) activity is a measure of the amount of sigma activity that is free per polymerase unit with poly d(A-T) as template. This ratio standardizes the different values of free sigma activity obtained from one experiment to another. The ratio te DNA activity/free a activity is equivalent to the ratio bound a activity/free a activity, since RNA polymerase activity with 'e DNA as template is a direct function of the amount of vegetative sigma factor that is bound to the core enzyme.
RESULTS Zone centrifugation analysis. Figure 1A and 1A' depict the results of zone centrifugation of an SI extract of vegetative cells. RNA polymerase sedimented as a major species with an S value of 14.5, and as a minor species with an S value of 13, when assayed with poly d(A-T) (Fig. 1A'). The minor species is probably a small amount of the 13S material, which is observed early in sporulation (see Fig. 1B' and C') and arises due to asynchrony of the cells. The maximal activity corresponded to a sedimentation coefficient of about 14.5S, in good agreement with values found by several authors (1, 10). The transcription activity with poly d(A-T) as template can be taken as an indication of total RNA polymerizing activity, whereas activity with the 'Ie DNA is a measure of sigma factor bound to the enzyme complex. It should be noted that by assaying sigma-factor activity throughout the gradient it was revealed that a large fraction of this factor is not bound to RNA polymerase and has a sedimentation coefficient of about 4S. Further, the RNA polymerase seems to be present only as holoenzyme (i.e., core enzyme is completely saturated with sigma factor), because its R:Mn value is less than 3 (2). This method of analysis was applied to several extracts prepared from cells harvested at t l1, to, t1, and t,. In Fig. 1 are
CY)
1.51
1
E N
2 1 2 A
T
~~~~~~~0.5~~~~~~~~~
U
1
2
I\~~~~~~~~~0 QL5-
5
10
1
205
10
15
20
Fracion number FIG. 1. Zone centrifugation of extracts of strain 168 harvested at different times. SI extracts (1 to 2 mg) of cells harvested at four different times were layered onto 12-ml linear gradients of 10 to 30% (vol/vol) glycerol in buffer containing 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.9, 0.01 M MgCl2, 0.1 M KCI, 0.0001 M ethylenediaminetetraacetate, and 0.0001 M dithiothreitol. After centrifugation for 15 h at 38,000 rpm in a Spinco-Beckman SW41 rotor at 4 C, fractions were collected with an Isco model 183 density gradient fractionator. (AA', BB', CC', and DD') Assays of extracts from cells harvested at t 1, to, t1, and t2, respectively. (A, B, C, and D) Vegetative sigma activities (A); 0.050 ml of each fraction was added at 0.2 ml of the standard transcription assay mixture which contained rifamycin (1 ug/ml, final concentration), Se DNA, and vegetative rifamycin-resistant core enzyme (1.5 polymerase units with poly d(A-T) as template) (2). Net stimulatory activity has been plotted after subtracting background activity of the added core polymerase with 4e DNA. (A', B', C', and D') Polymerase activities, 0.050 ml of each fraction, were assayed in the presence of poly d(A-T) (0) or $e DNA (0) under the standard conditions (2). The arrows show the position of beef liver catalase (UIS). The bottom of the gradient is on the right of the picture. 76
VOL. 125, 1976
EVOLUTION OF RNA POLYMERASE
represented the patterns of enzymatic activities detected after centrifugation; the results obtained are summarized in Table 1. Whereas for extracts prepared at any given time the measured activities varied from one experiment to another, depending on the amount of protein layered on the gradient, the ratio of activities was nearly constant. Thus, this method of analysis seems suitable for comparisons of polymerase activities of different extracts. Decrease of vegetative sigma activity. At every time point represented in Fig. 1, a large fraction of total sigma-factor activity was free (Fig. 1A-D). (Although the assay for free a factor was not expected to reveal any activity in the region of the gradient containing the RNA polymerase holoenzyme, some activity, representing about 15% of the total a factor present in this region, was measured. This indicates that a small proportion of the a factor bound to core enzyme can be displaced by the addition of vegetative rifamycin-resistant core enzyme.) Between to and t2 free sigma activity, 4S, rapidly decreased (Table 1). This observation is in good agreement with previous results (2). Decrease in association of sigma-factor and RNA polymerase activities. The values of the ratio $e DNA activity/free a activity also decreased as a function of time of extraction. This means that the fraction of sigma activity bound to the polymerase became smaller. This result was confirmed by the enhancement of ratio of the transcription complex: the ability of the enzyme complex to transcribe $e DNA decreased steadily relative to transcription of poly d(A-T) (2). It is particularly interesting to
77
compare the values obtained from extracts of t l cells and to cells. Between these two time points the R:Mn ratio increases, indicating that the to enzyme complex is less active than vegetative enzyme in transcribing $e DNA. At the same time, the fraction of sigma-factor activity bound to the enzyme decreased ($e DNA activity/free activity ratio), and the amount of free sigma per unit of polymerase increased. This decreased association of polymerase and sigmafactor activities becomes augmented after the to event, while total sigma activity is decreasing. This apparent loss of affinity was confirmed by examining a mixture of extracts of the strain M021 (RfmR) harvested at t-o., and the wildtype (Rfm5) strain harvested at t,. The t-o., extract contributes an excess of sigma and a vegetative polymerase whose activity is the only one measured in the mixture when assay tubes contain rifamycin. The activity of the t, enzyme was deduced by comparison of activities measured with and without rifamycin. This procedure is suitable if the polymerase activities are additive and if the RfmR enzyme is completely resistant to rifamycin. In fact, the additivity is generally good, but the resistance to the antibiotic of the M021 polymerase is only 90%. However, I have verified that, by mixing rifamycin-sensitive and -resistant polymerases with known activities and measuring these activites with and without antibiotic, the values obtained for the rifamycin-sensitive activities are close to the expected values (+10%; results not shown). In Fig. 2 is shown the pattern of activities of the individual extracts. The pattern of activities a
TABLE 1. 7ranscription activities during sporulation of strain 168 and at t7 for two SpoO strainsa Extract
d(A-T) activity
DNA Se activity
R:Mn
Free a
activity
Free a
te DNA
activity d(A-T)
activity/
activity
free a activity
168 t ,° 168 tob 168 toc 168 t1b 168 t25 168 t2c
9.3 13.1 2.0 12.2 5.8 18.8
4.5 4.5 0.7 1.4 0.4 1.2
2.0 2.9 2.9 8.5 13.1 15.6
14.8 26.4 3.5 10.8 3.5 8.6
1.6 2.0 1.8 0.9 0.6 0.5
0.30 0.17 0.20 0.13 0.11 0.14
SpoOb 6Z t4d MO 32 t4d
13.0 6.1
2.7 2.0
4.8 3.1
10.1 7.7
0.8 1.3
0.27 0.26
aThe values of activities obtained, expressed in polymerase units, represent the sum of the activities obtained in the different fractions. $e DNA activity, sum of the transcription activities obtained with 4e DNA as template; d(A-T) activity, sum of the activities obtained with poly d(A-T); free a activity, sum of the vegetative sigma activities unbound to RNA polymerase; R:Mn, poly d(A-T) activity/4e DNA activity (2). b Values obtained from the analysis presented in Fig. 1. c Values from similar analysis (i.e., Fig. 1) but not represented. d Values from analysis presented in Fig. 4.
78
J. BACTERIOL.
BREVET
T° 0.5
0
-o0.5erS 10
5
10
113
20
25
3
15
20
25
30
Fraction number FIG. 2. Zone centrifugation of extracts of cells harvested at t O. 6 and at t,l Same conditions as in Fig. 1. (A) Extract of strain M021 harvested at t-0.5; (B) extract of strain 168 harvested at t,. obtained with the mixture of these two extracts is shown in Fig. 3, and the results of these measurements are summarized in Table 2. In the mixture, the two polymerase activities (RfmR, t0., and RfmS, t1) are in the presence of the same amount of sigma activity, but the t1 enzyme has less active sigma factor bound to it than does the vegetative enzyme. Thus the transcription complex from sporulating cells behaves as if it has a lower affinity for the sigma
factor than does the corresponding complex derived from vegetative cells. Successive forms of transcription complex during sporulation. The patterns of polymerase activities obtained after zone centrifugation suggest an evolution of the transcription complex during the course of sporulation (Fig. 1). The major peak of polymerase activity of a vegetative extract sediments at 14.5S and is roughly symmetric and has transcriptional activity with both Se DNA and poly d(A-T); a more slowly sedimenting shoulder has little activity with the phage DNA as template. An extract of to or t1 cells, however, has two closely sedimented major peaks, with S values 13 and 14.5, neither of which is efficient in transcribing 4e DNA. To analyze more clearly the pattern of polymerase activities after zone centrifugation, a greater number of fractions was collected from gradients of cell extracts harvested at t-o.5
(RfmR) and t, (Rfms) (Fig. 2) or from mixture of these two extracts (Fig. 3). With the t-0.6 extract (Fig. 2A) a broad peak of polymerase activity was observed, having an S value of about 14.5, whereas with the t1 extract (Fig. 2B) two distinct peaks of activities were observed, with S values of 13 and 16. In the mixture of t 0. 5 and t, extracts, activity with 4e DNA gave only one peak, with an S value of 14.5, when rifamycin was included in the reaction mixture (Fig. 3B), whereas the rifamycin-sensitive activity, taken as the difference between the total activity, and the rifamycin-resistant activity exhibited two peaks (Fig. 3C) of about 13S and 16S. These values are identical to those found in the analysis of each extract alone. These changes in the S value of polymerase activity occur reproducibly, suggesting that the transcription complex exhibits structural changes as a function of time of its extraction. The 14.5S species, present in vegetative cells, is transformed into either a 13S species or a 16S species or both, these two latter species being present together at about t1. Analysis by SDS-gel electrophoresis of the ,B subunits. With respect to the 13S RNA polymerase, it is possible that partial degradation of the enzyme occurring in vitro (in spite of the presence of protease inhibitors) could be responsible for its appearance. This observed S value is compatible with the structure of proteolytically damaged RNA polymerase once thought to be present in sporulating cells (9, 10). The 13S species I obtained after zone centrifugation of a t1 extract was examined by polyacrylamide gel electrophoresis in the presence of SDS to see if any structural modification of the fl subunits could be detected. Figure 4 shows that no changes in the mobilities of the ,B polypeptides were observed in the 13S polymerase; the ,B subunits of this polymerase were indistinguishable from those of vegetative polymerase. Nothing can be concluded from this analysis concerning other subunits having molecular weights less than 100,000, since the enzyme preparation was impure. The most slowly migrating polypeptides are presumed not to be part of RNA polymerase. Transcription complex of asporogenous strain blocked at stage 0. Our analysis of the transcription complex was extended to two asporogenous mutant strains blocked at stage 0 of sporulation, SpoOb 6Z(RfmS) and M032(RfmRSpo-). The mutations involved mapping in widely separated chromosomal regions. The phenotypes of these two strains with respect to sporulation-associated functions are differ-
VOL. 125, 1976
ent, but neither is able to carry out change in RNA polymerase template specificity characteristic of wild-type cells after to (Table 3; 3, 19). Extracts were prepared from cells harvested at hour 4 after the end of logarithmic growth. The patterns of transcription activity after zone sedimentation are shown in Fig 5, and the values of the different activities and rates are included in Table 1. With strain M032, free sigma activity per polymerase unit (1.3) was close to the value found with vegetative wild cells. In a similar manner, the *e DNA activity/free ff activity ratio (0.26) was comparable to that of the vegetative extract (0.30) and high enough to suggest that the enzyme had not lost sigma affinity. The pattern of polymerase activity (Fig. 5B) was roughly symmetrical and had an S value of 14.5; little, if any, of the 13S or 16S species appeared. At to the transcription complex of this SpoO strain does not seem different from that found in vegetative cells. With an extract of strain SpoOb 6Z harvested at to (Table 1) it is more difficult to interpret the results. The free sigma activity per polymerase unit was low (0.8) and was similar to the value found with the wild-type strain harvested at t1 (0.9). But the R:Mn ratio (4.8) suggests a reasonable ability of the enzyme to transcribe *e DNA, and the ratio of $e DNA activity/free a activity (0.27) is close enough to the vegetative value (0.30) to suggest that the affinity of the complex for sigma is not seriously interfered with. One possible interpretation of these results is that the pool of active sigma is diminished by a mechanism that may be presumed to function in wild-type sporulating cells as well as in this mutant. Analysis of the polymerase activity pattern of the mutant is consistent with this explanation. An important fraction of the polymerase activity was present as the 13S species, whereas the 16S species was not detected. This pattern of polymerase activity looks like that found with an extract of the wild strain harvested at to (Fig. 1B'). Thus, strain SpoOb 6Z might carry out a partial evolution of the transcription complex.
DISCUSSION By using zone centrifugation of partially purified enzyme preparations it was possible to distinguish several protein complexes involved in transcription. The results obtained indicate that at any time, until at least t2, an excess of free sigma activity is present in the extracts, and that a variable fraction is bound to the core enzyme. Between to and t2, the free sigma
EVOLUTION OF RNA POLYMERASE
79
activity decreases as a function of time. This observation is in good agreement with our previous results (2). This decrease may be due to the destruction or inactivation of sigma factor or to the appearance of a specific inhibitor. Recently it was reported that crude extracts of t, cells lacking active sigma contain as much sigma polypeptide as extracts of vegetative cells, that sigma polypeptide in extracts from sporulating cells is apparently only weakly associated with RNA polymerase, and that sigma from sporulating cells is apparently unaltered and not intrinsically reduced in its ability to bind to core polymerase (22). The authors cited suggested that sporulating bacteria contain an inhibitor of sigma activity and that this inhibitor acts by interfering with the binding of the sigma subunit to RNA polymerase. It is interesting to note that Khesin et al. (7) and Stevens (21) have isolated fractions from E. coli infected with phage T4 that inhibit sigma activity in vitro. It would be interesting to search for a similar inhibitor of sigma activity during sporulation and, in this case, to know if the inhibitor is bound to sigma factor or to the core enzyme.
The *e DNA activity/free a activity ratio decreases as a function of time. This fact means that the complex of transcription has decreased association with active sigma. The appearance of an enzyme-bound inhibitor, as well as a chemical alteration of the vegetative core enzyme, could lead to this observation. This last possibiltiy seems unlikely, since $e DNA transcription by pure core enzyme extracted at t, is stimulated normally by addition of vegetative sigma factor (2) and since the electrophoretic mobilities of the t3 ', t, and a subunits are indistinguishable from those of vegetative enzyme (2, 9, 15). To confirm the decreased association of polymerase and active vegetative sigma factor after to, an extract of wild cells, harvested at tl, was mixed with an extract of rifamycin-resistant cells harvested in the log phase. This last extract was a source of sigma activity. The results showed that, in spite of this excess of free sigma activity, the t1 polymerase was much less able to bind this activity than was the vegetative enzyme. It thus appears that the transcription complex of sporulating cells has decreased affinity for sigma. The pattern of polymerase activity seen after zone centrifugation is in agreement with this hypothesis. It is seen that at different times of extraction three different species of polymerase complex appear. In the extract of vegetative
a 0
x
0 x
1.5
a-
E
U
CV
1.5
I I
-o In0
-a40
4A 0.5 a,
0.5 -Q u
5
10 15 20 25 Froction number
30
Froction number 80
VOL. 125, 1976
EVOLUTION OF RNA POLYMERASE
81
TABLE 2. Transcription activities of MO 21 t0o.5 and 168 t1 extractsa Extract
d(A-T)
activity
4'e DNA activity
R-Mn
M021 t0.5b
4.5
168 t1b
6.6
2.1 0.9
2.2 7.3
18.7 9.6
5.1 3.4
3.6 2.8
9.1
1.8
5.1
Free a activity
Free a
e DNA
activity/ d(A-T)
activity/ free a
activity
activity
0.6 1.1
0.47 0.31
1.2
0.16
Mixture of two above extractsc
Total activity Rifamycin-resistant activity (MO21 t-o.d Rifamycin-sensitive activity (168 t )
10.9
results are expressed as in Table 1. Values from the analysis represented in Fig. 2. c Values from the analysis represented in Fig. 3.
a The
cells a species having an S value of about 14.5S is present, whereas two others appear after t. and have values of 13S and 16S. In the mixture of t 0.5 and t1 extracts, three polymerase species can be separated. An evolution of the transcription complex, which can be schematically represented in the following manner, seems to be indicated:
13S species, 16S species 14.5S species tl vegetative phase to The appearance of the 13S species is not due to proteolytic cleavage of the # subunits. The loss of sigma factor bound to the core enzyme may be sufficient to explain the observed S value, but in addition the enzyme may change in conformation. The 16S polymerase could result from the association of new protein factors with the core enzyme in such a way that the apparent molecular weight would be modified. This hypothetical association is consistent with the results obtained by Greenleaf and collaborators (5) and Nishimoto and Takahashi (14), who found that at the beginning of sporulation new proteins become bound to the polymerase. The role played by these proteins is still unknown, and it is not yet possible to conclude whether the 16S polymerase is in fact associated with any of these proteins.
The scheme proposed above is suggested by the results obtained with wild-type cells harvested at different times. The analysis of some sporulation mutants serves to test it. The results obtained with two SpoO strains are not incompatible with the hypothesis. Mutant SpoOb 6Z harvested at to possesses two polymerase activities of 13S and 14.5S. The fact that the 16S species is absent enhances the idea that the 13S species appears earlier. The results of analysis of the extract from strain M032 harvested at to are nearly identical to those obtained with vegetative cell extracts, little if any loss of free sigma activity and little if any 13S species (cf. Fig. 5B and Fig. 1A'). The single mutation in a gene coding for RNA polymerase that confers resistance to rifamycin to the M032 enzyme could conceivably suppress any transformation of the 14.5S species of the transcription complex to the 13S species. In the case of strain SpoOb 6Z the primary structure of the polymerase subunits is not affected. Since a partial transformation of the polymerase occurs, the necessary functions must be present, at least in part. Furthermore, with strain SpoOb 6Z harvested after the end of growth, free sigma activity is low, whereas with strain M032 it is as high as in a vegetative cell extract. Thus we could think that the function that reduces free sigma activity is related to the appearance
FIG. 3. Zone centrifugation of a mixture of extracts of cells harvested at t_o. and at ti. The t_... cells are strain M021 (RfmR) and t, cells are strain 168 (Rfms). Ten polymerase units of transcription activity, with poly d(A-T) as template of each preparation, were mixed and layered onto the glycerol gradient. (a) Vegetative sigma activity as measured in Fig. 1; (A) total polymerase activities (assays without rifamycin); (B) resistant polymerase activities in the presence of 1 Aug of rifamycin per ml; (C) rifamycin-sensitive polymerase activities (by difference between total activities and rifamycin-resistant activities). Symbols: (0) polymerase activity with poly d(A-T); (0) polymerase activity with $e DNA; (A) vegetative sigma activity.
82
J. BACTERIOL.
BREVET
TABLE 3. Phenotype of the Spo- mutants Stage
Strain
0 0
(I n~~~~~~~~~~~~~~0
c'
R:Mn5 Est
age
Abs
0 0
-
+
-
-
+
+
ND
_
SpoOb 6Z M032 (rfm76)
0.5
Pheotypea Spr NarR
block-
2.7 1.3
a Sporulation-related phenotype designations are abbreviated as follows: antibiotic, serine-protease, and esterase production are Abs+, Spr+, and Est+, respectively; inducibility of nitrate reductase is NarR+ and constitutivity is NarR- (6, 12). ND, Not
done. bR:Mn, [specific activity with poly d(A-T) V (specific activity with be DNA) (2, 3).
0.5
2
2
4
6
8
10
Migration (cm,
FIG. 4. Densitometer tracing after SDS-gel electrophoresis of vegetative holoenzyme and 13S polymerase. Proteins were dissociated in 100 ,l of a mixture of 0.0675 M tris(hydroxymethyOaminomethanehydrochloride, pH 6.8, 3% SDS, 5% 2-mercaptoethanol and 10%o glycerol and incubated for 2 min in a boiling-water bath. After addition of 10 Al of a solution (100 mg/ml) of sodium iodoacetate and 5 isl of a 0.1% solution of bromophenol blue, the mixture was submitted to electrophoresis as described in Materials and Methods. (A) 6 gg of vegetative holoenzyme; (B) 13S polymerase; to 200 ul of the appropriate glycerol gradient fraction was added 20 pl of 50%o (wt/vol) trichloroacetic acid. After 15 min on ice, a precipitate was collected by centrifugation and washed with cold ethyl ether to remove trichloroacetic acid. Proteins were dissociated as described above.
of the 13S species. It should be noted that strain M032 produces the sporulation-associated antibiotic S-protease and esterase activities in wildtype amounts (Table 3; 19), whereas these activities are reduced or null in strain SpoOb 6Z (Table 3; 6, 12). Thus these three activities are not responsible for any of the changes in structure or specificity of RNA polymerase that we have observed. The system of analysis described here has suggested a working hypothesis concerning changes in the activity and organization of the transcription complex during sporulation. Ad-
n
A
cg
AA
\AA
10
3
o
15
20
A53
Al A'A
(A) StanSob Z(0.(BStanM3 O (a) poyease aciiywtBoydAI) lyers acitywh i$e NA 5
10
15
20
25
o
30
FIG. 5. Zone centrifugation of extracts of SpoO tp. Same conditions as in Fig. 1. strains harvested at (A) Strain SpoOb 6Z (Rfms). (B) Strain M032 oidct however,sigma ditioalwokis (A) vegetative activity; Symbols:ractiond (RfMRSpoo). (0) polymerase activity with poly d(A-T); (0) polymerase activity with 4Fe DNA.
ditional work is required, however, to indicate the physiological significance of the different species of RNA polymerase we have observed.
VOL. 125, 1976 ACKNOWLEDGMENTS I thank A. L. Sonenshein for helpful discussions and for assistance in preparing the manuscript, P. Schaeffer for helpful advice and support during the course of this work, and G. Vellard for expert technical assistance. This work was supported by grants to P. Schaeffer from the Centre National de la Recherche Scientifique (Laboratoire Associe 136), the Fondation from la Recherche Medicale Francaise, and the Commissariat a l'Energie Atomique.
LITERATURE CITED 1. Avila J., J. M. Hermoso, E. Vineula, and M. Salas. 1970. Subunit composition of Bacillus subtilis RNA polymerase. Nature (London) 226:1244-1245. 2. Brevet, J. 1974. Direct assay for sigma factor activity and demonstration of loss of this activity during sporulation in Bacillus subtilis. Mol. Gen. Genet. 128:223-231. 3. Brevet J., and A. L. Sonenshein. 1972. Template specificity of ribonucleic acid polymerase in asporogenous mutants of Bacillus subtilis. J. Bacteriol. 112:1270-1274. 4. Burgess, R. R. 1969. A new method for the large scale purification of Escherichia coli deoxyribonucleic aciddependent ribonucleic acid polymerase. J. Biol. Chem. 244:6160-6167. 5. Greenleaf A. L., T. G. Linn, and R. Losick. 1973. Isolation of a new RNA polymerase-binding protein from sporulating Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 70:490-494. 6. Ionesco H., J. Michel, B. Cami, and P. Schaeffer. 1970. Genetics of sporulation in Bacillus subtilis Marburg. J. Appl. Bacteriol. 33:13-24. 7. Khesin R. B., E. S. Bogdanova, A. D. Goldfarb, Jr., and Y. N. Zograff. 1972. Competition for the DNA template between RNA polymerase molecules from normal and phage-infected E. coli Mol. Gen. Genet. 119:299-314. 8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Linn T. G., A. L. Greenleaf, R. G. Shorenstein, and R. Losick. 1973. Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc. Natl. Acad. Sci. U.S.A. 70:1865-1869. 10. Losick, R., R. G. Shorenstein, and A. L. Sonenshein. 1970. Structural alteration of RNA polymerase during sporulation. Nature (London) 227:910-913.
EVOLUTION OF RNA POLYMERASE
83
11. Losick, R., and A. L. Sonenshein, 1969. Change in the template specificity of RNA polymerase during sporulation of Bacillus subtilis. Nature (London) 224:35-57. 12. Michel, J. F., and B. Cami. 1969. Selection de mutants de Bacillus bloques au debut de la sporulation. III. Nature des mutations selectionnees Ann. Inst. Pasteur Paris 116:3-18. 13. Milanesi, G., and J. Brevet. 1974. Template specificity of transcription during sporulation of Bacillus subtilis. Nucleic Acids Res. 1:397-412. 14. Nishimoto, H., and I. Takahashi. 1973. Subunits and template specificity of RNA polymerase from asporogenous mutants of B. subtilis. Colloq. Int. C.N.R.S. 227: 9-10. 15. Orrego, C., P. Kerjan, M. C. Manca De Nadra, and J. Szulmajster. 1973. Ribonucleic acid polymerase in a thermosensitive sporulation mutant (ts-4) of Bacillus subtilis. J. Bacteriol. 116:636-647. 16. Pene, J. J. 1969. Modification of transcription following interaction of template specific monomers of Bacillus subtilis RNA polymerase. Nature (London) 223:705-707. 17. Schaeffer, P., J. Millet, and J. P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. U.S.A. 54:704-711. 18. Shorenstein, R. G., and R. Losick. 1973. Purification and properties of the sigma subunit of ribonucleic acid polymerase from vegetative Bacillus subtilis. J. Biol. Chem. 248:6163-6169. 19. Sonenshein, A. L., B. Cami, J. Brevet, and R. Cote. 1974. Isolation and characterization of rifampin-resistant and streptolydigin-resistant mutants of Bacillus subtilis with altered sporulation properties. J. Bacteriol. 120:253-265. 20. Sonenshein, A. L., and R. Losick. 1970. RNA polymerase mutants blocked in sporulation. Nature (London) 227:906-909. 21. Stevens, A. 1974. Deoxyribonucleic acid dependent ribonucleic acid polymerases from two T4 phage-infected systems. Biochemistry 13:493-503. 22. Tjian, R., and R. Losick. 1974. An immunological assay for the sigma subunit of RNA polymerase in extracts of vegetative and sporulating Bacillus subtilis Proc. Natl. Acad. Sci. U.S.A. 71:2872-2876. 23. Travers, A., and R. Buckland. 1973. Heterogeneity of E. coli RNA polymerase. Nature (London) New Biol. 243:257-260.
JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 74-83 Copyright 0 1976 American Society for Microbiology
Evolution of the Transcription Complex During Sporulation of Bacillus subtilis JEAN BREVET' Institut de Microbiologie, Universite Paris-Sud, Centre d'Orsay, 91405 Orsay, France Received for publication 29 July 1975
Ribonucleic acid polymerase activity in partially purified extract of cells of Bacillus subtilis harvested at different times (t 1, to, t,, and t2) was studied by zone centrifugation. During the course of sporulation, vegetative sigma-factor activity decreased and the transcription complex lost some of its affinity for active sigma factor. The complex underwent a two-stage change in sedimentation value, from 14.5S in vegetative growth phase to a 13S species very early in sporulation to a 16S species at later times. Two Spo0 mutants have been studied by zone centrifugation. One strain, a rifampin-resistant (RfmR) mutant, failed to show any modification of the transcription complex, whereas the other, a Rfms strain, underwent a partial evolution of the transcription complex after to.
The change in template specificity of ribonu- a t2 extract and separated from the holoenzyme cleic acid (RNA) polymerase, which takes place by diethylaminoethyl-cellulose chromatograduring the first 2 h of sporulation (t0-t2) (10), is phy. correlated with loss of vegetative sigma-factor Through the use of zone centrifugation techactivity (2, 9). Since highly purified core en- niques, it has been possible to demonstrate zyme extracted from cells at t, in the presence physical and functional heterogeneity of RNA of protease inhibitors has the same subunit polymerase in crude extracts of growing cells of composition as vegetative core enzyme (2, 9, 15) Bacillus subtilis (16) and Escherichia coli (23). and responds to the addition of vegetative In the present work, I have looked for possible sigma factor, sporulation core seems to be changes in the transcription system during indistinguishable from vegetative core enzyme. sporulation using zone centrifugation analyses These facts and the recent results of Tjian and of partially purified extracts of cells harvested Losick (22) suggest that interference with bind- at t-l, to, tl, and t2. I show here that during ing or activity of the vegetative sigma factor sporulation there is a decrease in the affinity of rather than structural modification of vegeta- RNA polymerase for the vegetative sigma factor tive core enzyme subunits is responsible for the and that there is a changing pattern in the sedimentation coefficient of the transcription change in template specificity. Many single-site mutations conferring resist- complex. ance to rifamycin derivatives or streptolydigin to the core enzyme affect at the same time the MATERIALS AND METHODS ability to sporulate and the change in specificity Bacterial strains. Spo+ strains used were 168 of the polymerase. The favored interpretation is (trpC2) and M021 (RfmR) (2), and Spo- strains were that transcription of sporulation genes requires SpoOb 6Z (6, 12) and M032 (RfmR, Spoo) (19). changes in the enzyme which may no longer be Bacterial cultures. Bacteria were cultivated at possible when this enzyme is altered by muta- 37 C with vigorous rotary agitation in 2-liter Fernbach tion. Such changes may be alterations in the flasks containing 500 ml of nutrient broth medium enzyme structure or binding of specific factors described by Schaeffer et al. (17). Growth was followed of transcription. These putative factors might turbidometrically at 570 nm with a Zeiss spectrophotometer. Under these conditions, at t. (8 h after the be lost during the course of purification of RNA end of exponential growth) 80% of the cells present at polymerase. One such case was previously de- the beginning of the stationary phase were resistant to scribed (13): a protein fraction that stimulated heating for 10 min at 80 C. t,, of sporulation refers to n transcription of coliphage T7 deoxyribonucleic hours after the end of exponential growth. acid (DNA) preferentially was demonstrated in Buffers. The buffers used are derived from those described by Burgess (4). Buffer G contained 0.05
I Present address: Division of Clinical Pharmacology, Department of Medicine, Stanford University Medical School, Stanford, Calif. 94305.
M
Tris(hydroxymethyl)aminomethane-hydrochloride,
pH 7.9; 0.01 M MgCl,; 0.2 M KCl; 0.1 mM 74
VOL. 125, 1976
ethylenediaminetetraacetate; 0.1 mM dithiothreitol; 10% (vol/vol) glycerol. Buffer A contained 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.9; 0.1 M KCl; 0.1 mM ethylenediaminetetraacetate; 0.1 mM dithiothreitol; stated glycerol concentration. Preparation of celi extracts. One liter of a vegetative culture or 500 ml of stationary-phase culture was centrifuged (15,000 x g for 10 min at +4 C), and the cells (ca. 1 g wet weight) were washed once in buffer G, quickly frozen, and stored at - 70 C until used. Pellets were suspended in 10 ml of buffer G, to which inhibitors of B. subtilis proteases had been added: phenylmethylsulfonyl fluoride (0.5 ml of an alcoholic solution at 12 mg/ml) and ethylenediaminetetraacetate-Mg-Na, (5 mM) (protease inhibitors prepared just before use). The pH was adjusted by the addition of 1 N KOH. The cells were broken by two 2-min exposures to sonication (MSE desintegrator), keeping temperature below 4 C. Ribosomes were removed by centrifugation for 90 min at 100,000 x g. Proteins in the supernatant fluid were precipitated by the addition of 2.1 g of ammonium sulfate per 10 ml, and the precipitate was removed by centrifugation at 100,000 x g for 30 min. Then, 2.1 g of ammonium sulfate per 10 ml was added to the supernatant fluid to give a 60% saturated solution. The precipitate was centrifuged at 100,000 x g for 30 min, and the pellet (SI extract) was dissolved in 1 to 2 ml of buffer A without glycerol. A fraction of this extract was withdrawn for zone centrifugation analysis, and to the remainder an equal volume of buffer A containing 90% glycerol (vol/vol) was added and the extract was stored at - 20 C. Zone sedimentation analysis. One to 2 mg of protein from an SI extract in buffer A without glycerol was immediately applied to a 12-ml linear gradient of 10 to 30% (vol/vol) glycerol in buffer A and centrifuged for 15 h at 38,000 rpm in a Spinco SW41 rotor. Fractions were collected, and 50 Al was assayed for sigma activity or RNA polymerase (Fig. 1, legend). Polymerase assay. The RNA polymerase assay has been described previously (3), with the following modifications: assays using $e DNA as template were done in the presence of 10 mM MgCl,, whereas assays using poly d(A-T) were done in the presence of 1 mM MnCl, (2). ['H ]adenosine 5'-triphosphate was used at 6,000 counts/min per nmol. Sigma activity. The assay conditions for sigma activity were identical to those described above for RNA polymerase activity with $e DNA as template, except that 2 ;&g of pure vegetative core enzyme extracted from strain M021 (RfmR) and rifamycin (1 /ug/ml, a concentration inhibitory to the wild-type enzyme) were added (2). The presence of rifamycin eliminated the contribution of the RNA polymerase activity present in the fractions of the gradient. SDS-polyacrylamide gel electrophoresis. Conditions for sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis were according to Laemmli (8), the polyacrylamide gels (12 cm) being prepared according to Linn et al. (9). Expression of the results. One unit of RNA polymerase activity is defined as the amount of
EVOLUTION OF RNA POLYMERASE
75
enzyme that incorporates 1 nmol of ['H Jadenosine 5'-monophosphate in 10 min at 37 C. One unit of net stimulatory sigma activit& increased the activity of excess vegetative core polymerase with te DNA by one RNA polymerase unit. In analyzing glycerol gradients, values for RNA polymerase activity in the presence of poly d(A-T), RNA polymerase activity in the presence of te DNA, and free vegetative sigma activity were the sums of the activities found in the appropriate fractions of the gradients. From these values several ratios were calculated. R:Mn is the ratio of transcription on poly d(A-T) to transcription on te DNA (2, 3) and is a measure of the template specificity of the enzyme since *e DNA serves as template for only RNA polymerase holoenzyme, whereas poly d(A-T) is transcribed by both holoenzyme and core enzyme. Previous experiments indicated that the activities of vegetative holoenzyme on these substrates produced a R:Mn ratio of 2 to 3 (unpublished data; 2). The ratio free a activity/ d(A-T) activity is a measure of the amount of sigma activity that is free per polymerase unit with poly d(A-T) as template. This ratio standardizes the different values of free sigma activity obtained from one experiment to another. The ratio te DNA activity/free a activity is equivalent to the ratio bound a activity/free a activity, since RNA polymerase activity with 'e DNA as template is a direct function of the amount of vegetative sigma factor that is bound to the core enzyme.
RESULTS Zone centrifugation analysis. Figure 1A and 1A' depict the results of zone centrifugation of an SI extract of vegetative cells. RNA polymerase sedimented as a major species with an S value of 14.5, and as a minor species with an S value of 13, when assayed with poly d(A-T) (Fig. 1A'). The minor species is probably a small amount of the 13S material, which is observed early in sporulation (see Fig. 1B' and C') and arises due to asynchrony of the cells. The maximal activity corresponded to a sedimentation coefficient of about 14.5S, in good agreement with values found by several authors (1, 10). The transcription activity with poly d(A-T) as template can be taken as an indication of total RNA polymerizing activity, whereas activity with the 'Ie DNA is a measure of sigma factor bound to the enzyme complex. It should be noted that by assaying sigma-factor activity throughout the gradient it was revealed that a large fraction of this factor is not bound to RNA polymerase and has a sedimentation coefficient of about 4S. Further, the RNA polymerase seems to be present only as holoenzyme (i.e., core enzyme is completely saturated with sigma factor), because its R:Mn value is less than 3 (2). This method of analysis was applied to several extracts prepared from cells harvested at t l1, to, t1, and t,. In Fig. 1 are
CY)
1.51
1
E N
2 1 2 A
T
~~~~~~~0.5~~~~~~~~~
U
1
2
I\~~~~~~~~~0 QL5-
5
10
1
205
10
15
20
Fracion number FIG. 1. Zone centrifugation of extracts of strain 168 harvested at different times. SI extracts (1 to 2 mg) of cells harvested at four different times were layered onto 12-ml linear gradients of 10 to 30% (vol/vol) glycerol in buffer containing 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.9, 0.01 M MgCl2, 0.1 M KCI, 0.0001 M ethylenediaminetetraacetate, and 0.0001 M dithiothreitol. After centrifugation for 15 h at 38,000 rpm in a Spinco-Beckman SW41 rotor at 4 C, fractions were collected with an Isco model 183 density gradient fractionator. (AA', BB', CC', and DD') Assays of extracts from cells harvested at t 1, to, t1, and t2, respectively. (A, B, C, and D) Vegetative sigma activities (A); 0.050 ml of each fraction was added at 0.2 ml of the standard transcription assay mixture which contained rifamycin (1 ug/ml, final concentration), Se DNA, and vegetative rifamycin-resistant core enzyme (1.5 polymerase units with poly d(A-T) as template) (2). Net stimulatory activity has been plotted after subtracting background activity of the added core polymerase with 4e DNA. (A', B', C', and D') Polymerase activities, 0.050 ml of each fraction, were assayed in the presence of poly d(A-T) (0) or $e DNA (0) under the standard conditions (2). The arrows show the position of beef liver catalase (UIS). The bottom of the gradient is on the right of the picture. 76
VOL. 125, 1976
EVOLUTION OF RNA POLYMERASE
represented the patterns of enzymatic activities detected after centrifugation; the results obtained are summarized in Table 1. Whereas for extracts prepared at any given time the measured activities varied from one experiment to another, depending on the amount of protein layered on the gradient, the ratio of activities was nearly constant. Thus, this method of analysis seems suitable for comparisons of polymerase activities of different extracts. Decrease of vegetative sigma activity. At every time point represented in Fig. 1, a large fraction of total sigma-factor activity was free (Fig. 1A-D). (Although the assay for free a factor was not expected to reveal any activity in the region of the gradient containing the RNA polymerase holoenzyme, some activity, representing about 15% of the total a factor present in this region, was measured. This indicates that a small proportion of the a factor bound to core enzyme can be displaced by the addition of vegetative rifamycin-resistant core enzyme.) Between to and t2 free sigma activity, 4S, rapidly decreased (Table 1). This observation is in good agreement with previous results (2). Decrease in association of sigma-factor and RNA polymerase activities. The values of the ratio $e DNA activity/free a activity also decreased as a function of time of extraction. This means that the fraction of sigma activity bound to the polymerase became smaller. This result was confirmed by the enhancement of ratio of the transcription complex: the ability of the enzyme complex to transcribe $e DNA decreased steadily relative to transcription of poly d(A-T) (2). It is particularly interesting to
77
compare the values obtained from extracts of t l cells and to cells. Between these two time points the R:Mn ratio increases, indicating that the to enzyme complex is less active than vegetative enzyme in transcribing $e DNA. At the same time, the fraction of sigma-factor activity bound to the enzyme decreased ($e DNA activity/free activity ratio), and the amount of free sigma per unit of polymerase increased. This decreased association of polymerase and sigmafactor activities becomes augmented after the to event, while total sigma activity is decreasing. This apparent loss of affinity was confirmed by examining a mixture of extracts of the strain M021 (RfmR) harvested at t-o., and the wildtype (Rfm5) strain harvested at t,. The t-o., extract contributes an excess of sigma and a vegetative polymerase whose activity is the only one measured in the mixture when assay tubes contain rifamycin. The activity of the t, enzyme was deduced by comparison of activities measured with and without rifamycin. This procedure is suitable if the polymerase activities are additive and if the RfmR enzyme is completely resistant to rifamycin. In fact, the additivity is generally good, but the resistance to the antibiotic of the M021 polymerase is only 90%. However, I have verified that, by mixing rifamycin-sensitive and -resistant polymerases with known activities and measuring these activites with and without antibiotic, the values obtained for the rifamycin-sensitive activities are close to the expected values (+10%; results not shown). In Fig. 2 is shown the pattern of activities of the individual extracts. The pattern of activities a
TABLE 1. 7ranscription activities during sporulation of strain 168 and at t7 for two SpoO strainsa Extract
d(A-T) activity
DNA Se activity
R:Mn
Free a
activity
Free a
te DNA
activity d(A-T)
activity/
activity
free a activity
168 t ,° 168 tob 168 toc 168 t1b 168 t25 168 t2c
9.3 13.1 2.0 12.2 5.8 18.8
4.5 4.5 0.7 1.4 0.4 1.2
2.0 2.9 2.9 8.5 13.1 15.6
14.8 26.4 3.5 10.8 3.5 8.6
1.6 2.0 1.8 0.9 0.6 0.5
0.30 0.17 0.20 0.13 0.11 0.14
SpoOb 6Z t4d MO 32 t4d
13.0 6.1
2.7 2.0
4.8 3.1
10.1 7.7
0.8 1.3
0.27 0.26
aThe values of activities obtained, expressed in polymerase units, represent the sum of the activities obtained in the different fractions. $e DNA activity, sum of the transcription activities obtained with 4e DNA as template; d(A-T) activity, sum of the activities obtained with poly d(A-T); free a activity, sum of the vegetative sigma activities unbound to RNA polymerase; R:Mn, poly d(A-T) activity/4e DNA activity (2). b Values obtained from the analysis presented in Fig. 1. c Values from similar analysis (i.e., Fig. 1) but not represented. d Values from analysis presented in Fig. 4.
78
J. BACTERIOL.
BREVET
T° 0.5
0
-o0.5erS 10
5
10
113
20
25
3
15
20
25
30
Fraction number FIG. 2. Zone centrifugation of extracts of cells harvested at t O. 6 and at t,l Same conditions as in Fig. 1. (A) Extract of strain M021 harvested at t-0.5; (B) extract of strain 168 harvested at t,. obtained with the mixture of these two extracts is shown in Fig. 3, and the results of these measurements are summarized in Table 2. In the mixture, the two polymerase activities (RfmR, t0., and RfmS, t1) are in the presence of the same amount of sigma activity, but the t1 enzyme has less active sigma factor bound to it than does the vegetative enzyme. Thus the transcription complex from sporulating cells behaves as if it has a lower affinity for the sigma
factor than does the corresponding complex derived from vegetative cells. Successive forms of transcription complex during sporulation. The patterns of polymerase activities obtained after zone centrifugation suggest an evolution of the transcription complex during the course of sporulation (Fig. 1). The major peak of polymerase activity of a vegetative extract sediments at 14.5S and is roughly symmetric and has transcriptional activity with both Se DNA and poly d(A-T); a more slowly sedimenting shoulder has little activity with the phage DNA as template. An extract of to or t1 cells, however, has two closely sedimented major peaks, with S values 13 and 14.5, neither of which is efficient in transcribing 4e DNA. To analyze more clearly the pattern of polymerase activities after zone centrifugation, a greater number of fractions was collected from gradients of cell extracts harvested at t-o.5
(RfmR) and t, (Rfms) (Fig. 2) or from mixture of these two extracts (Fig. 3). With the t-0.6 extract (Fig. 2A) a broad peak of polymerase activity was observed, having an S value of about 14.5, whereas with the t1 extract (Fig. 2B) two distinct peaks of activities were observed, with S values of 13 and 16. In the mixture of t 0. 5 and t, extracts, activity with 4e DNA gave only one peak, with an S value of 14.5, when rifamycin was included in the reaction mixture (Fig. 3B), whereas the rifamycin-sensitive activity, taken as the difference between the total activity, and the rifamycin-resistant activity exhibited two peaks (Fig. 3C) of about 13S and 16S. These values are identical to those found in the analysis of each extract alone. These changes in the S value of polymerase activity occur reproducibly, suggesting that the transcription complex exhibits structural changes as a function of time of its extraction. The 14.5S species, present in vegetative cells, is transformed into either a 13S species or a 16S species or both, these two latter species being present together at about t1. Analysis by SDS-gel electrophoresis of the ,B subunits. With respect to the 13S RNA polymerase, it is possible that partial degradation of the enzyme occurring in vitro (in spite of the presence of protease inhibitors) could be responsible for its appearance. This observed S value is compatible with the structure of proteolytically damaged RNA polymerase once thought to be present in sporulating cells (9, 10). The 13S species I obtained after zone centrifugation of a t1 extract was examined by polyacrylamide gel electrophoresis in the presence of SDS to see if any structural modification of the fl subunits could be detected. Figure 4 shows that no changes in the mobilities of the ,B polypeptides were observed in the 13S polymerase; the ,B subunits of this polymerase were indistinguishable from those of vegetative polymerase. Nothing can be concluded from this analysis concerning other subunits having molecular weights less than 100,000, since the enzyme preparation was impure. The most slowly migrating polypeptides are presumed not to be part of RNA polymerase. Transcription complex of asporogenous strain blocked at stage 0. Our analysis of the transcription complex was extended to two asporogenous mutant strains blocked at stage 0 of sporulation, SpoOb 6Z(RfmS) and M032(RfmRSpo-). The mutations involved mapping in widely separated chromosomal regions. The phenotypes of these two strains with respect to sporulation-associated functions are differ-
VOL. 125, 1976
ent, but neither is able to carry out change in RNA polymerase template specificity characteristic of wild-type cells after to (Table 3; 3, 19). Extracts were prepared from cells harvested at hour 4 after the end of logarithmic growth. The patterns of transcription activity after zone sedimentation are shown in Fig 5, and the values of the different activities and rates are included in Table 1. With strain M032, free sigma activity per polymerase unit (1.3) was close to the value found with vegetative wild cells. In a similar manner, the *e DNA activity/free ff activity ratio (0.26) was comparable to that of the vegetative extract (0.30) and high enough to suggest that the enzyme had not lost sigma affinity. The pattern of polymerase activity (Fig. 5B) was roughly symmetrical and had an S value of 14.5; little, if any, of the 13S or 16S species appeared. At to the transcription complex of this SpoO strain does not seem different from that found in vegetative cells. With an extract of strain SpoOb 6Z harvested at to (Table 1) it is more difficult to interpret the results. The free sigma activity per polymerase unit was low (0.8) and was similar to the value found with the wild-type strain harvested at t1 (0.9). But the R:Mn ratio (4.8) suggests a reasonable ability of the enzyme to transcribe *e DNA, and the ratio of $e DNA activity/free a activity (0.27) is close enough to the vegetative value (0.30) to suggest that the affinity of the complex for sigma is not seriously interfered with. One possible interpretation of these results is that the pool of active sigma is diminished by a mechanism that may be presumed to function in wild-type sporulating cells as well as in this mutant. Analysis of the polymerase activity pattern of the mutant is consistent with this explanation. An important fraction of the polymerase activity was present as the 13S species, whereas the 16S species was not detected. This pattern of polymerase activity looks like that found with an extract of the wild strain harvested at to (Fig. 1B'). Thus, strain SpoOb 6Z might carry out a partial evolution of the transcription complex.
DISCUSSION By using zone centrifugation of partially purified enzyme preparations it was possible to distinguish several protein complexes involved in transcription. The results obtained indicate that at any time, until at least t2, an excess of free sigma activity is present in the extracts, and that a variable fraction is bound to the core enzyme. Between to and t2, the free sigma
EVOLUTION OF RNA POLYMERASE
79
activity decreases as a function of time. This observation is in good agreement with our previous results (2). This decrease may be due to the destruction or inactivation of sigma factor or to the appearance of a specific inhibitor. Recently it was reported that crude extracts of t, cells lacking active sigma contain as much sigma polypeptide as extracts of vegetative cells, that sigma polypeptide in extracts from sporulating cells is apparently only weakly associated with RNA polymerase, and that sigma from sporulating cells is apparently unaltered and not intrinsically reduced in its ability to bind to core polymerase (22). The authors cited suggested that sporulating bacteria contain an inhibitor of sigma activity and that this inhibitor acts by interfering with the binding of the sigma subunit to RNA polymerase. It is interesting to note that Khesin et al. (7) and Stevens (21) have isolated fractions from E. coli infected with phage T4 that inhibit sigma activity in vitro. It would be interesting to search for a similar inhibitor of sigma activity during sporulation and, in this case, to know if the inhibitor is bound to sigma factor or to the core enzyme.
The *e DNA activity/free a activity ratio decreases as a function of time. This fact means that the complex of transcription has decreased association with active sigma. The appearance of an enzyme-bound inhibitor, as well as a chemical alteration of the vegetative core enzyme, could lead to this observation. This last possibiltiy seems unlikely, since $e DNA transcription by pure core enzyme extracted at t, is stimulated normally by addition of vegetative sigma factor (2) and since the electrophoretic mobilities of the t3 ', t, and a subunits are indistinguishable from those of vegetative enzyme (2, 9, 15). To confirm the decreased association of polymerase and active vegetative sigma factor after to, an extract of wild cells, harvested at tl, was mixed with an extract of rifamycin-resistant cells harvested in the log phase. This last extract was a source of sigma activity. The results showed that, in spite of this excess of free sigma activity, the t1 polymerase was much less able to bind this activity than was the vegetative enzyme. It thus appears that the transcription complex of sporulating cells has decreased affinity for sigma. The pattern of polymerase activity seen after zone centrifugation is in agreement with this hypothesis. It is seen that at different times of extraction three different species of polymerase complex appear. In the extract of vegetative
a 0
x
0 x
1.5
a-
E
U
CV
1.5
I I
-o In0
-a40
4A 0.5 a,
0.5 -Q u
5
10 15 20 25 Froction number
30
Froction number 80
VOL. 125, 1976
EVOLUTION OF RNA POLYMERASE
81
TABLE 2. Transcription activities of MO 21 t0o.5 and 168 t1 extractsa Extract
d(A-T)
activity
4'e DNA activity
R-Mn
M021 t0.5b
4.5
168 t1b
6.6
2.1 0.9
2.2 7.3
18.7 9.6
5.1 3.4
3.6 2.8
9.1
1.8
5.1
Free a activity
Free a
e DNA
activity/ d(A-T)
activity/ free a
activity
activity
0.6 1.1
0.47 0.31
1.2
0.16
Mixture of two above extractsc
Total activity Rifamycin-resistant activity (MO21 t-o.d Rifamycin-sensitive activity (168 t )
10.9
results are expressed as in Table 1. Values from the analysis represented in Fig. 2. c Values from the analysis represented in Fig. 3.
a The
cells a species having an S value of about 14.5S is present, whereas two others appear after t. and have values of 13S and 16S. In the mixture of t 0.5 and t1 extracts, three polymerase species can be separated. An evolution of the transcription complex, which can be schematically represented in the following manner, seems to be indicated:
13S species, 16S species 14.5S species tl vegetative phase to The appearance of the 13S species is not due to proteolytic cleavage of the # subunits. The loss of sigma factor bound to the core enzyme may be sufficient to explain the observed S value, but in addition the enzyme may change in conformation. The 16S polymerase could result from the association of new protein factors with the core enzyme in such a way that the apparent molecular weight would be modified. This hypothetical association is consistent with the results obtained by Greenleaf and collaborators (5) and Nishimoto and Takahashi (14), who found that at the beginning of sporulation new proteins become bound to the polymerase. The role played by these proteins is still unknown, and it is not yet possible to conclude whether the 16S polymerase is in fact associated with any of these proteins.
The scheme proposed above is suggested by the results obtained with wild-type cells harvested at different times. The analysis of some sporulation mutants serves to test it. The results obtained with two SpoO strains are not incompatible with the hypothesis. Mutant SpoOb 6Z harvested at to possesses two polymerase activities of 13S and 14.5S. The fact that the 16S species is absent enhances the idea that the 13S species appears earlier. The results of analysis of the extract from strain M032 harvested at to are nearly identical to those obtained with vegetative cell extracts, little if any loss of free sigma activity and little if any 13S species (cf. Fig. 5B and Fig. 1A'). The single mutation in a gene coding for RNA polymerase that confers resistance to rifamycin to the M032 enzyme could conceivably suppress any transformation of the 14.5S species of the transcription complex to the 13S species. In the case of strain SpoOb 6Z the primary structure of the polymerase subunits is not affected. Since a partial transformation of the polymerase occurs, the necessary functions must be present, at least in part. Furthermore, with strain SpoOb 6Z harvested after the end of growth, free sigma activity is low, whereas with strain M032 it is as high as in a vegetative cell extract. Thus we could think that the function that reduces free sigma activity is related to the appearance
FIG. 3. Zone centrifugation of a mixture of extracts of cells harvested at t_o. and at ti. The t_... cells are strain M021 (RfmR) and t, cells are strain 168 (Rfms). Ten polymerase units of transcription activity, with poly d(A-T) as template of each preparation, were mixed and layered onto the glycerol gradient. (a) Vegetative sigma activity as measured in Fig. 1; (A) total polymerase activities (assays without rifamycin); (B) resistant polymerase activities in the presence of 1 Aug of rifamycin per ml; (C) rifamycin-sensitive polymerase activities (by difference between total activities and rifamycin-resistant activities). Symbols: (0) polymerase activity with poly d(A-T); (0) polymerase activity with $e DNA; (A) vegetative sigma activity.
82
J. BACTERIOL.
BREVET
TABLE 3. Phenotype of the Spo- mutants Stage
Strain
0 0
(I n~~~~~~~~~~~~~~0
c'
R:Mn5 Est
age
Abs
0 0
-
+
-
-
+
+
ND
_
SpoOb 6Z M032 (rfm76)
0.5
Pheotypea Spr NarR
block-
2.7 1.3
a Sporulation-related phenotype designations are abbreviated as follows: antibiotic, serine-protease, and esterase production are Abs+, Spr+, and Est+, respectively; inducibility of nitrate reductase is NarR+ and constitutivity is NarR- (6, 12). ND, Not
done. bR:Mn, [specific activity with poly d(A-T) V (specific activity with be DNA) (2, 3).
0.5
2
2
4
6
8
10
Migration (cm,
FIG. 4. Densitometer tracing after SDS-gel electrophoresis of vegetative holoenzyme and 13S polymerase. Proteins were dissociated in 100 ,l of a mixture of 0.0675 M tris(hydroxymethyOaminomethanehydrochloride, pH 6.8, 3% SDS, 5% 2-mercaptoethanol and 10%o glycerol and incubated for 2 min in a boiling-water bath. After addition of 10 Al of a solution (100 mg/ml) of sodium iodoacetate and 5 isl of a 0.1% solution of bromophenol blue, the mixture was submitted to electrophoresis as described in Materials and Methods. (A) 6 gg of vegetative holoenzyme; (B) 13S polymerase; to 200 ul of the appropriate glycerol gradient fraction was added 20 pl of 50%o (wt/vol) trichloroacetic acid. After 15 min on ice, a precipitate was collected by centrifugation and washed with cold ethyl ether to remove trichloroacetic acid. Proteins were dissociated as described above.
of the 13S species. It should be noted that strain M032 produces the sporulation-associated antibiotic S-protease and esterase activities in wildtype amounts (Table 3; 19), whereas these activities are reduced or null in strain SpoOb 6Z (Table 3; 6, 12). Thus these three activities are not responsible for any of the changes in structure or specificity of RNA polymerase that we have observed. The system of analysis described here has suggested a working hypothesis concerning changes in the activity and organization of the transcription complex during sporulation. Ad-
n
A
cg
AA
\AA
10
3
o
15
20
A53
Al A'A
(A) StanSob Z(0.(BStanM3 O (a) poyease aciiywtBoydAI) lyers acitywh i$e NA 5
10
15
20
25
o
30
FIG. 5. Zone centrifugation of extracts of SpoO tp. Same conditions as in Fig. 1. strains harvested at (A) Strain SpoOb 6Z (Rfms). (B) Strain M032 oidct however,sigma ditioalwokis (A) vegetative activity; Symbols:ractiond (RfMRSpoo). (0) polymerase activity with poly d(A-T); (0) polymerase activity with 4Fe DNA.
ditional work is required, however, to indicate the physiological significance of the different species of RNA polymerase we have observed.
VOL. 125, 1976 ACKNOWLEDGMENTS I thank A. L. Sonenshein for helpful discussions and for assistance in preparing the manuscript, P. Schaeffer for helpful advice and support during the course of this work, and G. Vellard for expert technical assistance. This work was supported by grants to P. Schaeffer from the Centre National de la Recherche Scientifique (Laboratoire Associe 136), the Fondation from la Recherche Medicale Francaise, and the Commissariat a l'Energie Atomique.
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EVOLUTION OF RNA POLYMERASE
83
11. Losick, R., and A. L. Sonenshein, 1969. Change in the template specificity of RNA polymerase during sporulation of Bacillus subtilis. Nature (London) 224:35-57. 12. Michel, J. F., and B. Cami. 1969. Selection de mutants de Bacillus bloques au debut de la sporulation. III. Nature des mutations selectionnees Ann. Inst. Pasteur Paris 116:3-18. 13. Milanesi, G., and J. Brevet. 1974. Template specificity of transcription during sporulation of Bacillus subtilis. Nucleic Acids Res. 1:397-412. 14. Nishimoto, H., and I. Takahashi. 1973. Subunits and template specificity of RNA polymerase from asporogenous mutants of B. subtilis. Colloq. Int. C.N.R.S. 227: 9-10. 15. Orrego, C., P. Kerjan, M. C. Manca De Nadra, and J. Szulmajster. 1973. Ribonucleic acid polymerase in a thermosensitive sporulation mutant (ts-4) of Bacillus subtilis. J. Bacteriol. 116:636-647. 16. Pene, J. J. 1969. Modification of transcription following interaction of template specific monomers of Bacillus subtilis RNA polymerase. Nature (London) 223:705-707. 17. Schaeffer, P., J. Millet, and J. P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. U.S.A. 54:704-711. 18. Shorenstein, R. G., and R. Losick. 1973. Purification and properties of the sigma subunit of ribonucleic acid polymerase from vegetative Bacillus subtilis. J. Biol. Chem. 248:6163-6169. 19. Sonenshein, A. L., B. Cami, J. Brevet, and R. Cote. 1974. Isolation and characterization of rifampin-resistant and streptolydigin-resistant mutants of Bacillus subtilis with altered sporulation properties. J. Bacteriol. 120:253-265. 20. Sonenshein, A. L., and R. Losick. 1970. RNA polymerase mutants blocked in sporulation. Nature (London) 227:906-909. 21. Stevens, A. 1974. Deoxyribonucleic acid dependent ribonucleic acid polymerases from two T4 phage-infected systems. Biochemistry 13:493-503. 22. Tjian, R., and R. Losick. 1974. An immunological assay for the sigma subunit of RNA polymerase in extracts of vegetative and sporulating Bacillus subtilis Proc. Natl. Acad. Sci. U.S.A. 71:2872-2876. 23. Travers, A., and R. Buckland. 1973. Heterogeneity of E. coli RNA polymerase. Nature (London) New Biol. 243:257-260.
