Developmental and genetic regulation of bacillus subtilis genes transcribed by σ28-RNA polymerase

Cell, Vol . 35, 285 -293, November 1983, Copyright t 1983 by MIT

0092-8674/83/110285-09 $020010

Developmental and Genetic Regulation of Bacillus subtilis Genes Transcribed by a"-RNA Polymerase

Michael Z . Gilman* and Michael J . Chamberlin Department of Biochemistry University of California Berkeley, California 94720

Summary Sigma-28 RNA polymerase is a minor form of Bacillus subtilis RNA polymerase that is highly specific for transcription from a small number of promoter sites in the B. subtilis genome . We have followed transcription from two of these loci (P 28-1 and P28-2) in vivo using a quantitative S 1 nuclease mapping procedure. Both promoters are used at a modest rate in vegetatively growing cells (about 10 RNA copies per cell) and transcripts are initiated at the same start sites as found in vitro with the purified 0-28 -RNA polymerase. Transcription from the o 28 promoters varies somewhat with growth conditions and is shut off rapidly and almost completely after the first hour of sporulation . Neither a28 transcripts is detected in vegetative cells of certain B . subtilis mutants (spoO classes A, B, E, and F) that are defective in sporulation . Transcription from these promoters is restored in second site revertants that are able to sporulate . Hence the action of o28-RNA polymerase appears to be regulated by the spoO genes and the functions controlled by o28-promoters may be closely tied to the system involved in the initiation of sporulation . Introduction Bacillus subtilis possesses multiple forms of RNA polymerase with differing promoter specificities (for review see Losick and Pero, 1981 ; Doi, 1982a, 1982b). All forms share in common the core subunits, /3', /3, and a, while each contains a unique sigma factor . It is the sigma factor that confers the characteristic promoter recognition specificity on each RNA polymerase holoenzyme . Five different cellular forms of RNA polymerase have been isolated from B . subtilis . 0-55 -RNA polymerase, the predominant form, is homologous to E . coli r 70 -RNA polymerase (Shorenstein and Losick, 1973a, 1973b) and efficiently reads E . coli promoters (Wiggs et al ., 1979) . Minor forms of B . subtilis RNA polymerase include v37- ( Haldenwang and Losick, 1979, 1980), 0 32 - (Johnson et al ., 1983), and o 28 -RNA polymerases (Jaehning et al ., 1979; Wiggs et al ., 1981) which are all present at low levels in vegetatively growing cells, and o 29 -RNA polymerase which has been identified in sporulating cells (Haldenwang et al ., 1981) . What role do these minor forms of RNA polymerase play in B . subtilis growth and development? Losick and Pero (1981) suggest that they are important in the control of Present address : Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 .

sporulation . In vitro both 0-32- and 0-37 -RNA polymerases transcribe genes that are expressed during sporulation . These polymerases persist in early sporulating cells, and Losick and his collaborators have shown that transcription of the spoVG gene, which is read by both a37- and a32RNA polymerases, is induced in the early stages of sporulation . Transcription of spoVG is dependent on at least seven of the known spoO genes, which control the earliest stages of sporulation (Ollington et al ., 1981 ; Zuber and Losick, 1983) . In addition, at least one new sigma factor, r 29 , appears as sporulation progresses . In contrast, 0-28-RNA polymerase has not previously been linked to sporulation, and its function remains unknown . It was first detected in a partially purified preparation of RNA polymerase from vegetatively growing B . subtilis as an RNA polymerase activity that had an in vitro promoter recognition specificity distinct from any of the known bacterial RNA polymerases including the 0-55 enzyme (Jaehning et al ., 1979) . This activity was subsequently separated from 0- 55-RNA polymerase by heparin-agarose chromatography, and the specificity of the enzyme was shown to be the result of the presence of a new sigma factor with an apparent molecular weight of 28,000 daltons (Wiggs et al ., 1981) . One of the most striking properties of 0-28 -RNA polymerase is its stringent template preference : it is virtually inert on templates lacking cognate promoters (Wiggs et al ., 1981 ; Gilman et al ., 1981) . This stringency allowed us to isolate two B . subtilis genomic fragments that carry 0- 28 promoters . These promoters are read very efficiently in vitro by r28-RNA polymerase and contain strong sequence homologies (Gilman et al ., 1981) . We now estimate that the B . subtilis genome carries no more than 20-30 strong promoters for 0- 28-RNA polymerase . We have cloned a number of these sites (Gilman et al ., submitted), but the identities of the genes they control are unknown . In order to determine the function of r 28 -RNA polymerase and the set of chromosomal genes it controls, we have investigated the expression of two of these genes in vivo . We show here that both loci are expressed in vivo and that the 5' ends of these RNAs map precisely to the 0- 28 RNA polymerase in vitro start sites previously determined (Gilman et al ., 1981) . We have used a quantitative S1 nuclease protection assay to measure the levels of these RNAs under a variety of growth conditions and in different B . subtilis strains . We find that these transcripts are both nutritionally regulated during vegetative growth and temporally regulated during development . Their expression depends on the products of several genes that control sporulation . The functions encoded in 0-28-controlled genes may participate in the molecular network that senses nutrient deprivation and triggers spore development in B . subtilis . Results 0-28 -RNA Polymerase In Vivo Transcription Units We originally isolated two B. subtilis chromosomal fragments that carry strong in vitro promoter sites for o 28 -RNA

Cell 286

polymerase as Pst I fragments in the vector pHV 14 (Wiggs et al., 1981). Subclones of these fragments were then isolated in pBR322, giving plasmids pMGlO2 and pMG201 (Gilman et al., 1981). Restriction maps of these fragments together with the location and orientation of the d?s-specific promoters are shown in Figure 1, To investigate the in vivo expression of these loci, we initially used a blot hybridization procedure (Thomas, 1980) to identify in vivo RNAs that hybridized to these cloned fragments. One of these fragments (pMG201, carrying promoter P,& hybridizes to a 1.6 kb RNA found in vegetatively growing B. subtilis (Figure 2). This transcript is identical in size to the in vitro transcript obtained by transcription of pCD4322 plasmid DNA with purified $‘-RNA polymerase, which terminates at an efficient in vitro termination site contained within the cloned B. subtilis DNA (Wiggs et al., 1981). As we show below, the 5’ ends of the in vivo and in vitro transcripts are identical. Although we have not yet mapped the 3’ ends of these transcripts, they are likely to be close to or identical to those found in vitro. It appears, therefore, that the insert in pCD4322 carries a complete transcription unit for P-RNA polymerase. In preliminary studies we were unable to detect RNA hybridizing to the other 0” promoter-containing fragment (pMG102, carrying promoter PZ8.,, see Figure 1) on blots. Therefore we turned to the more sensitive procedure of Sl nuclease mapping (Berk and Sharp, 1977; Weaver and Weissman, 1979). With this technique, we clearly identified RNAs arising from both chromosomal r?* promoters in vegetatively growing B. subtilis (Figure 3). The 5’ ends of these in vivo RNAs were identical, at nucleotide resolution, to those obtained in vitro with purified components. The abundance of both in vivo transcripts, estimated by reference to a standard curve prepared with known amounts of in vitro RNA, is approximately ten copies per cell during log phase growth in nutrient sporulation medium (NSM). We conclude that both uz8 promoters are used in vivo and

that the initiation specificity identical in vivo and in vitro.

of g*-RNA

polymerase

is

Sl Nuclease Protection Assay for In Vivo Transcripts Because Sl nuclease protection allowed us to detect in vivo $a transcripts sensitively and specifically, we investigated whether we could use this technique quantitatively to measure the abundance of these transcripts under a variety of growth conditions. Figure 4 shows a reconstitution experiment in which increasing amounts of in vitro $8RNA polymerase transcripts were analyzed by the Sl technique in the presence of a constant amount of carrier RNA. No specific probe fragments were protected in the presence of carrier RNA alone. With increasing amounts of in vitro RNA, we observed an increasing amount of protection of a specific fragment of the same size obtained with in vivo RNA. When these bands were excised and the radioactivity was counted and plotted against the amount of specific RNA in the hybridization mixture, a linear relationship was obtained through at least 6 fmole of transcript (Figure 4). The Sl nuclease protection assay is quantitative, extremely sensitive, and can detect RNAs present in amounts well below one copy per cell. Most significantly for these studies, it is absolutely promoter-specific and any change in the site of transcript initiation, arising perhaps from the activation of closely-linked promoters, can be easily detected. This is a significant advantage over techniques that follow expression using gene fusions, for example. Fur-

123

23s

4

-

16s17 2.2

PCD4322

P28-2

1 R

PMGPOI

% 4.,

0.75 -

TI

, Aic

1.6

R

Probe Figure 2. Northern Blot Hybridization of B. subtilis RNA Read from Promoter 262.RNAs (25 Ag each) Prepared from Cells Growing in Different Media Were Glyoxalated, and Separated by Gel Electrophoresis Figure 1. Restriction

Maps of 8. subtilis Promoter-Containing

Fragments

The positions and orientations of the promoters are indicated, Below each map are the restriction fragments used as hybridization probes for Sl nuclease protection expenments. Sites marked below the lines are restnction endonuclease sites: Ps: Pst I; EC: Eco RI; E’: Eco RI’; P: Pvu II; Hd: Hand III; Hp: Hpa II; Ha: Hae Ill. Sites marked above the line: Pa, $8 promoter, Ps5, 0% promoter; T: termination site.

Bands were then transferred to nitrocellulose and hybridized to nicktranslated pMG201 DNA as described in Thomas (1990). Growth media were: lane 1: super-rich, 3% glucose; lane 2: super-rich, 3% glycerol; lane 3: NSM, 0.09% glucose; lane 4: NSM. 0.09% glycerol. Positions of 16s and 23s rRNAs, determined by hybridrzatron to a cloned E. coli rrnB operon (pKK3535; Brosius et al., 1981) are indicated. Other markers are glyoxalated fragments of pBR322 DNA.

$alopmental

and Genetrc Regulation of Bacillus subtilrs

A P,8-1 12345

‘.

12345

nating at upstream promoter sites as long as controls are carried out to ensure that probe is used in excess. In the experiments that follow, we use this assay to measure the abundance of a’-‘-specific transcripts under a variety of conditions. As controls for these experiments, we have also assayed for the abundance of a typical transcript generated by the major form of 6. subtilis RNA polymerase, u~~-RNA polymerase. The promoter we used is the veg promoter isolated and characterized by Losick’s group (Ollington et al., 1981; Ollington and Losick, 1981; Moran et al., 1982) (Figure 1). In Figure 3, we show that Sl nuclease mapping confirms that vegetatively growing cells contain large amounts of an RNA mapping to the same site observed in vitro with purified u~~-RNA polymerase (Moran et al., 1982). This site corresponds to the lower bandset in the in vivo and in vitro lanes. The broad upper bandset visible most clearly in the in vitro samples but also present in in vivo RNA assays (see Figures 6 and 7) corresponds to the start site predicted for an RNA polymerase molecule bound at the upstream polymerase binding site found by LeGrice and Sonenshein (1982). These workers were unable to detect initiation at this site in vitro in a runoff transcription assay. We hesitate to ascribe these bands to 5’ ends of authentic transcripts with certainty, because this region is extremely AT-rich and may simply represent an Sl -sensitive site in longer hybrids. We have not characterized these species further.

‘28-2

12345678

Nutritional Regulation of a” Transcripts

Figure 3. High Resolution Sl N&ease Mapping of 6. subtilts In Vivo RNA and ?-RNA Polymerase In Vitro Transcription Products Procedures for Sl nuciease mapprng are outfined in Experimental Procedures. Samples were analyzed by electrophoresis on 8% polyacrylamideurea sequencrng gels. (A): Sl mapping of Pm., transcripts. Lane 1: 26 rg carrier tRNA; lane 2: “G+A” sequence ladder; lane 3: “C+T” sequence ladder; lane 4: 20 pg carrier tRNA and RNA transcribed in vitro by P-RNA polymerase using Eco RI-digested pMG102 DNA as template; lane 5: 20 pg rn vivo RNA prepared from B. subtilis WI68 growtng vegetatively in NSM. (6): Sl mapping of PZb2 transcripts. Lane 1: 20 rg carrier WNA; lane 2: “G+A” sequence ladder; lane 3: ‘C+T” sequence ladder; lane 4: 20 pg carrier tRNA and RNA transcribed in vitro by g-RNA polymerase using Hrnd Ill-digested pMG201 DNA as template; lane 5: 20 pg in vivo RNA prepared from B. subtilis W168 growing vegetatively in NSM. (C): Si mapping of veg transcripts. Lane I: ‘G” sequence ladder; lane 2: “G+A sequence ladder; lane 3: “C+r’ sequence ladder; lane 4: “c” sequence ladder; lane 5: 20 pg in vivo RNA prepared from B. subtilis W168 growing vegetatively in NSM; lane 6: RNA transcribed in vitro by &RNA polymerase usrng super-coiled ~213-1 DNA as template; lane 7: RNA transcribed in vitro by a--RNA polymerase using Barn HI-digested ~213-1 DNA as template.

thermore, unlike some procedures which amount of RNA hybridizing to a particular this assay is not compromised by RNA opposite DNA strand or by read-through

simply follow the region of DNA, copied from the transcripts origi-

As we have shown, B. subtilis cells growing vegetatively in a nutrient broth-based sporulation medium contain about ten copies of each ds transcript. We determined the level of these transcripts in cells growing in a variety of different media. Table 1 shows that cells growing vegetatively in a minimal medium containing amino acids as the sole carbon source contained approximately 5fold less of both 2’ transcripts than cells growing in nutrient sporulation medium (NSM), when equal amounts of RNA were assayed. Addition to the minimal medium of galactose, a nonmetabolizable sugar in B. subtilis, resulted in no change in the level of u*' transcripts, In contrast, addition of the metabolizable carbon sources glucose and glycerol caused a 2- 3-fold increase in these transcripts. These levels went up another 2-fold in a rich sporulation medium (NSM), but in a very rich medium containing 3% glucose, in which the cells do not sporulate, the levels of both $* transcripts declined 2-fold from the levels in NSM. We also investigated the effect of brief starvation of cells for ammonia (nitrogen) or phosphate on levels of the uz8 transcripts. B. subtilis WI 68 cells were grown in minimal medium with glucose as a carbon source for these experiments. No significant changes were noted during the course of a 30 min starvation followed by refeeding with excess nutrient (data not shown). The levels of ua transcripts were also unaffected by changes in the growth temperature of W168 cells over the range from 30°C to 49°C (data not shown).

Cdl

288

+A

23456709

1

P

No RNA

B. Subtilis RNA (in viva)

Cz8-RP

I1

fmol Figure 4. Quantitative

P2s-, Transcript

Si Nuclease

Protection

5--C?

fi

Transcripts

(in vitro) 7

8

9

L 10

11

12

13

,

fmol P2a-+ Transcript

Assay

Increasing amounts of RNA transcribed in vitro by PRNA polymerase using the indicated templates were hybridized to Si probes in the presence of 20 pg carrier tRNA. Sl protected fragments were analyzed by electrophoresis in 6% polyactylamide, 6.3 M urea gels (upper panels). After electophoresis and autoradiography, the 32P-labeled protected fragments were located in the gel, excised, and counted in a liquid scintillation counter. The recovered radioactivity was plotted against the amount of in vitro transcribed RNA in the hybridization reaction (lower panels). Small backgrounds have been subtracted. Upper left: Sl nuclease protection assay of Pa., transcripts. Lane 1: untreated hybridization probe, 10% of the amount used in each hybridization; lane 2: 20 ag carrier tRNA only; lanes 3 and 4: 20 rg each of 6. subtilis in VIVO RNA, lanes 5-9: 20 pg carrier tRNA and 0.6, 1.2, 3, 6, and 12 fmole, respectively, of in vitro transcripts (?-RNA polymerase, Eco RI-digested pMG102 DNA). Upper right: Sl nuclease protection assay of P,transcripts. Lane 1: untreated hybridization probe, 10% of the amount used in each hybridization; lanes 2-4: 20 pg carrier tRNA only; lanes 5 and 6: 20 fig B. subtilis in vivo RNA; lanes 7-13: 20 pg carrier tRNA and 0.33, 0.66, 1.3, 3.3. 6.6, 13, and 33 fmole, respectively, in vitro transcripts (P-RNA polymerase, Hind Ill-digested pMG201 DNA). Lower left: plot of probe protected as a function of input RNA in the experiment shown in the upper left panel. Lower right: plot of probe protected as a function of input RNA in the experiment shown In the upper right panel.

Thus the abundance of these (Y~’ transcripts is moderately regulated in response to specific nutritional conditions. In general, the level of these transcripts increased as the quality of the medium improved and the growth rate of the cells increased. Only in a very rich medium, in which sporulation is repressed, did we observe a decline in transcript abundance and under no conditions was severe repression of apa transcripts seen.

Developmental Regulation of a*’ Transcripts In response to depletion of specific nutrients ment, B. subtilis initiates an orderly program tion that culminates in the development endospore (Piggot and Coote, 1976; Hoch,

in its environof differentiaof a dormant 1976; Losick,

1982). There is a large body of evidence that suggests that changes in transcriptional machinery accompany sporulation (reviewed in Doi, 1977; Sonenshein and Campbell, 1978) and it has been suggested that changes in RNA polymerase, specifically in the sigma subunits, might in part regulate the temporal program of sporulation (Losick and Pero, 1981). With these notions in mind, we have examined the fate of the 4’ transcripts in sporulating cells. Figure 5 shows that both uz8 transcripts behaved identically in sporulating cells. There was a rapid decline in transcript abundance once sporulation began. Both transcripts were reduced 3- 4-fold in abundance after 1 hr of sporulation. They were virtually undetectable after 2-hr and remained absent through the fourth hour.



Developmental and Genetic Regulation of Bacillus subtilis 289

Table 1 . Expression of a' Transcripts in Different Growth Media Media

P28_1

P28-2

Minimal/amino acids

0 .20

0 .20

Min ./a . a . + galactose

0 .23

0 .16

Min ./a . a . + glycerol

0.63

0 .50

Min ./a . a . + glucose

0.57

0 .43

Nutrient sporulation

1 .00

1 .00

Super-rich (3% glucose)

0.41

0 .60

For growth in minimal media, W168 spores were allowed to germinate overnight in minimal medium containing 0 .5% casamino acids medium . 1 .5 ml of this overnight culture was used to inoculate 150 ml of prewarmed minimal/amino acids medium . This culture was grown at 37°C with vigorous shaking to Aeon = 0 .2 then divided into aliquots in prewarmed flasks containing the indicated carbon sources at a final concentration of 10 mM . At A. = 1, cells were harvested . For growth in rich media, 25 ml cultures were directly inoculated with spores and grown at 37°C to A . = 1 . RNA extraction and quantitative S1 nuclease protection assays were carried out as described in Experimental Procedures. Values given here are in arbitrary units relative to the level of expression in nutrient sporulation medium which is set as 1 .0 .

A t .0

I

E 0 .8~,

E!

N

n

P55-veg 1

0 .6

`

0.4 1 O 0`

.~ CAP

O

0.2

-1

P28-1,2 \ \

0

1

A-

A

3

4

Hours of Sporulotion Figure 5 . Abundance of P281, P282, and veg Transcripts during Sporulation A 100 ml culture of B . subtilis W168 was grown in nutrient sporulation medium with vigorous shaking at 37°C . At the indicated intervals, 10 ml aliquots were withdrawn and rapidly chilled . RNA extraction and quantitative 51 nuclease protection assays were performed as described in Experimental Procedures . Key to symbols : circles, P28-, transcripts ; boxes, P2&2 transcripts ; triangles, P,~~,transcripts ; vertical dotted line, To, the beginning of sporulation as defined by the sharp drop in exponential growth rate .

This immediate decline was specific for these a28 transcripts, because the a55 -specific veg transcript behaved differently . It persisted at vegetative levels through the first hour of sporulation . By 2 hr, it declined about 8-fold and remained at this new level through 4 hr of sporulation . We observed no change in the apparent 5' end of this transcript . This persisting veg-transcribing activity may be because of a low level of a55-RNA polymerase activity remaining in sporulating cells or to the activity of another form of RNA polymerase using the same start site and promoter recognition sequences . It has been observed

that purified preparations of a37 RNA polymerase can transcribe from the veg promoter at a low level (R . Losick, personal communication) . Thus we find that in addition to being nutritionally regulated in vegetatively growing cells, the a 28 transcripts are rather dramatically regulated during development . They rapidly and specifically disappear with the onset of sporulation . Expression of a 28 Transcripts in Sporulation Mutants Sporulation requires the action of at least fifty different genes (Piggot and Coote, 1976) . Several of these genes, designated spoO, are considered to be regulatory loci because mutants altered in these genes fail to initiate sporulation and in some cases fail to express a variety of sporulation-associated functions (Piggot and Coote, 1976 ; Hoch, 1976) . Ollington et al . (1981) and Zuber and Losick (1983) have reported that transcription of the sporulation gene spoVG, which is transcribed in vitro by a37 - (Moran et al ., 1981) and a32-RNA polymerases (Johnson et al ., 1983), is under the control of several spoO genes . They found that cells bearing mutations in spoOA, B, C, E, F, H, and K are deficient in transcription of this gene when cells are transferred to sporulation medium . We find that the a28 transcripts are under similar, though not identical, regulation by spoO gene products . In Figure 6, we present S1 nuclease protection assays of RNA extracted from a variety of spoO mutants growing vegetatively in NSM . Both a28 transcripts were present in strains W168 and JH642 which are wild-type for sporulation (lanes 3 and 4), but they were substantially reduced in strains bearing mutations in spoOA, E, B, and F (lanes 5-8, respectively) . In a spoOH mutant, which fails to synthesize spoVG RNA (Ollington et al ., 1981), both a28 transcripts were present at wild-type levels . This result is specific for the a28 transcripts ; the a55-specific veg transcript was found in similar levels in all strains (Figure 6C) . We also tested mutants in spoO genes J and K (strains 1S26 and 1S28 ; te Table 2) ; these both contain normal levels of both transcripts . To investigate the role of spoO genes in the control of sporulation, Sharrock and Leighton (unpublished data) have isolated a collection of spoO mutants carrying extragenic suppressors that restore sporulation to near wildtype levels . We have assayed RNA prepared from a number of these strains for the presence of a 28 - and specific transcripts . Figure 7 shows that both a28 transcripts were again present in the spot strains JH642 and W168 (lanes 3 and 10), reduced in a spoOF mutant (lane 4), and restored to wild-type levels in three spoOF mutants carrying independently isolated extragenic suppressors designated rvt2, 5, and 11 (lanes 5-7) . The best characterized of these mutations, rvt11, suppresses mutations in several different spoO genes, including spoOB . Lanes 8 and 9 in Figure 7 show that introduction of the rvt11 allele into a spoOB mutant also restored both a28 transcripts . a

55-

a

Cell 290

Table 2. Bacillus subtilis Strains Used in this Study

A. P28- I

Strain

Genotype

Source

w168

Wild-type

T. Leighton

JH642

trpC2 phe-I

J. Hoch

JH646

JH642 spoOAl2

JH647

JH642 s/xOEl

J. Hoch 1

J. Hoch

JH648

JH642 spoOBl36

J. Hoch

JH649

JH642 spoOF221

J. Hoch

JH651

JH642 spoOH61

J. Hoch

1 S26

spoOJ87

T. Leighton

1S28

spoOK141

trpC2 phe-I

T. Leighton

RS2120

spoOF221

rvt2 lys-1

T. Leighton

RS2130

spoOF221

rvt5 lys-I

T. Leighton

RS412

spoOF221

rvtl 1 Iys-1

T. Leighton

RS5011

spoOB136

rvtll

T. Leighton

trpC2 phe-I

lys-1 phe-1

lation is not merely fortuitous nor simply a reflection of general metabolic imbalance in spo0 mutants. Strains that are prevented from sporulating by mutations in the transcriptional and translational machinery of the cell, which apparently act by upsetting general cell physiology (Sharrock and Leighton, 1981; Wayne et al., 1981; Wayne and Leighton, 1981) do not affect 2’ transcripts (data not shown). The only mutants that lack 2’ transcripts are those with lesions in the specific sporulation control system of B. subtilis.

8. P28-2

Discussion

C. P55veg

123456789 Figure 6. Sl Nuclease spo0 Mutants

Protection

Assay of RNA Prepared

from B. Subtilis

(A): Pm., transcripts; (8): Pzs2 transcripts: (C): veg transcripts. Lane 1: untreated probe, 100/o of the amount used in each hybridization; lane 2: 20 pg carrier tRNA (in (A), slightly contaminated with material from the adjacent lane); lane 3: WI 68 (spa’); lane 4: JH642 (spa+); lane 5: JH646 (spoOA12); lane 6: JH647 (spcOEl1); lane 7: JH648 (spoOB136); lane 8: JH649 (spoOF221); lane 9: JH651 (spoOH81).

Again this form of regulation is specific for the P transcripts; the veg transcript was unaffected by any of these mutations. Thus mutations that restore the ability to sporulate to spo0 mutants also restore u28 transcripts. These results are quite intriguing for they suggest a close relationship between the elements that control P transcripts and those that control sporulation. The precise relationship between sporulation and the expression of u*’ transcripts is not yet clear, but we believe that this corre-

We investigated the in vivo expression of two B. subtilis chromosomal loci under the control of $*-RNA polymerase. We found that these loci are transcribed in vivo and that these in vivo transcripts map precisely to the 2’ promoter sites we previously mapped using in vitro techniques (Wiggs et al., 1981; Gilman et al., 1981). These transcripts are modestly regulated by nutritional conditions during vegetative growth. In general, expression of these loci increased as the quality of the growth medium improved in media that allow sporulation. In a very rich medium that suppresses sporulation, we observed decreased levels of the g2’ transcripts. The transcripts are dramatically regulated during development; as sporulation begins, both transcripts disappear rapidly and specifically. Both cr’* transcripts are strikingly reduced in strains carrying mutations in the regulatory genes spoOA, 8, E, and F but not spoOH J or K. Second site mutations that restore the sporulation defects in several of these mutants restore wild-type levels of both 2’ transcripts. Hence synthesis of the two r?’ transcripts we studied is controlled by at least four spo0 gene products, which are also needed for cells to enter the earliest stages of sporulation. We considered two general mechanisms by which the spo0 gene products might regulate 2’ transcripts. First, the spo0 products could modulate the activity of uz8, either

Developmental 291

and Genetic

Regulation of Bacillus subtilis

c. P 55veg

12345678

910

Figure 7. Sl Nuclease ProtectIon Assay of RNA Prepared spu0 Mutants and s&w+ Pseudorevertants

from B. subtilis

(A): Pa.. transcripts; (B): PpB2 transcripts; (C): veg transcripts. Lane 1: untreated probe, loo/o of the amount used in each hybridization; lane 2: 20 rg carrier tRNA; lane 3: JH642 (spa’); lane 4: JH649 (spoOF221); lane 5: RS2120 (spoOF221 rVr2); lane 6: RS2130 (spoOF221 wf5); lane 7: RS2112 (spoOF221 rVrl1); lane 6: JH648 (spoOB136): lane 9: RS5011 (spoOB136 rvtll); lane IO: W166 (spa’).

by controlling its synthesis or breakdown or through the action of a specific d aa inhibitor. Second, the spo0 gene products could exert their control directly on the 02’ promoters, modulating the level of transcription of these loci by repression or activation without affecting the activity of u’~-RNA polymerase itself. A similar model has been proposed to explain spo0 regulation of spoVG transcription (Moran et al., 1981; Losick, 1982). These two models differ in their predictions of the level of >‘-RNA polymerase activity detectable in extracts of spo0 cells. Preliminary studies have been carried out measuring a2’ RNA polymerase levels in the spoOA mutant and during the first two hours of sporulation (J. L. Wiggs, 1981 Ph.D.

thesis, University of California, Berkeley, California). These studies employed a rapid, one-step purification of total RNA polymerase from B. subtilis using affinity chromatography on heparin-agarose columns (Chamberlin et al., 1983). The activity of uz8 RNA polymerase was assayed by runoff transcription from DNA of plasmids pMG201 and pMG102 that had been cleaved by appropriate restriction endonucleases. Because of the high levels of endogenous u55 RNA polymerase a true quantitative analysis was not possible. However, levels of 4’ RNA polymerase appeared unchanged in the spoOA cells, while there was a significant reduction in transcripts from both 2’ promoters in extracts prepared from cells harvested 1 and 2 hr after the beginning of sporulation. The results with the spoOA mutant, taken with our current studies which show that transcription from g*’ promoters Ppe., and P28.2 depends on spoOA function, suggest that u28 is present in spoOA cells but that d?*RNA polymerase is blocked in its action. This somewhat favors the second hypothesis, above, in which the spo0 function is needed to activate the d?* promoters. This was unexpected since a28 promoters are read well by $%NA polymerase in vitro in the absence of additional factors, and it may be that the spoOA product is involved in blocking action of a repressor, for example. However, the first hypothesis is not completely ruled out since an inhibitor of gee function would behave quite similarly to such a repressor. Quantitative studies of the activity and state of c2’ RNA polymerase in extracts will be needed to resolve these questions. In so far as the two 028 transcripts we assayed are representative of all u2’ transcripts, these results allow us to draw several tentative conclusions concerning the function of o~~-RNA polymerase in B. subtilis growth and development and the identity of the cr2’-controlled genes. First, because the 2’ transcripts are substantially reduced (at least 5 to IO-fold) in some spo0 mutants and these strains still grow adequately in the vegetative state, the functions encoded by these two ti*-controlled genes are not essential for vegetative growth or are required only in small amounts. We emphasize that spo0 mutants are not absolutely normal with respect to vegetative growth; at least fifty polypeptides are missing in vegetative spoOA cells (Brehm et al., 1975; Linn and Losick, 1976). If spoOA mutants fail to synthesize all of the transcripts read from a*’ promoters on the B. subtilis genome (some 20-30 sites as estimated by Gilman et al., submitted), as well as some u37- and u32-specific transcripts (Ollington et al., 1981; Zuber et al., 1983), this alone would account for most of the proteins missing in these cells. In addition, because 2’ transcripts decline rapidly in abundance with the onset of sporulation, the functions encoded in these transcripts are likely to be gratuitous once the cells become committed to development. In support of this notion, Ferrari et al. (1982) have prepared a chromosomal deletion in the 1.6 kb transcription unit controlled by Pz8.p. Mutants carrying this deletion still spor-

Cell 29 2

ulated at wild-type levels in rich media . This suggests that the P28-2 gene product does not play an essential role in sporulation, but this is not a rigorous conclusion since the coding limits of this gene are not known, and the deletion, which removed 300 by from the 3' end of the transcription unit, may not have inactivated the encoded polypeptide . If the . 028 -controlled functions are not required for the vegetative phase or the sporulation phase, what is their role? One possibility suggested by these results is that their critical function is in the transition from the vegetative to the sporulation phase . We note that mutants that are impaired in this transition, the spoOs, fail to make 0. 28 transcripts, Second-site mutations that restore to cells the ability to make this transition also restore 0.28 transcripts . We have not found conditions or mutants that can successfully make this transition in the absence of 0.21 transcripts . We will show elsewhere that the B . subtilis chromosome probably contains no more than 20-30 strong 0. 28 promoter sites (Gilman et al ., submitted) . We propose that these loci may encode functions that participate in the detection of nutrient deprivation and/or the transduction of this information to the apparatus that triggers sporulation . For example, these genes may code for a family of surface receptor proteins that bind specific nutrients and send signals to the cytoplasm when unoccupied . The abundance of such receptors would need to be modulated as the growth rate changes to maintain appropriate receptor density on the cell surface, in much the same fashion as 28 the 0 . transcripts are regulated . Such receptors would no longer be required once the cells become committed to sporulation, as is seen with the 0 .28 transcripts . Loss by mutation of any single receptor gene might not obviously affect the sporulation phenotype under most conditions, but loss of all receptors by mutation in the spoO genes or in the gene encoding 0.28 would produce a cell unable to sporulate . Moreover, a mutant lacking a whole family of membrane proteins might also be expected to show gross surface abnormalities, a phenotype characteristic of many spoO mutants (Hoch, 1976 ; Hoch et al ., 1978) . We are addressing this working hypothesis in several ways . We have cloned a number of new B . subtilis chromosomal loci carrying o28 promoters (Gilman et al ., submitted) . We plan to characterize the in vivo expression of these loci to determine whether they behave similarly to the transcripts described in this study . By mapping these loci genetically and constructing deletions in them, we hope to identify their functions . Experimental Procedures Strains, Plasmids, and Media The B . subtilis strains used in this study are listed Table 2 . Plasmids pMG102 and pMG201 have been previously described (Gilman et al ., 1981) . They carry B . subtilis genomic fragments which contain o-" promoters . Plasmid p213-1 (Ollington and Losick, 1981), which carries the veg promoter, was generously provided by R . Losick (Harvard University) . We used three different growth media in these studies . Nutrient sporulation medium (Schaeffer et al ., 1965) contained 1 .6% Difco nutrient broth,

25 mM KCI, 2 mM CaCl2 , 10 -5 FeSO4, 10'4 M MgSO4 , 10 -5 M MnC12 , 0 .09% glucose. "Super-rich" medium contained 2% bacto-tryptone, 1% yeast extract, 21 mM Na2HPO4 , 3% glucose. The minimal medium was a modification of the MOPS-buffered medium of Neidhardt et al . (1974) (C . Price, personal communication) . It generally contained 50 mM MOPS (pH 7), 4 mM Tricine, 9 .5 mM NH 4C1, 0.52 MM MgC1 2 , 0.276 mM K2S04 , 0 .02 mM FeSO4 , 1 mM CaCl 2, 0.1 mM MnC12, 1 .32 mM K2SO4 and micronutrients at the concentration recommended by Neidhardt et al . (1974). Supplements and changes in this medium for individual experiments are detailed in the appropriate figure legends . Each growth experiment is described in detail in the relevant figure legend. Generally, 10 ml aliquots were withdrawn from log-phase cultures (A€ =1) and transferred to 50 ml centrifuge tubes containing ice chilled to -20°C . Cells were sedimented at 0°C and resuspended in 0 .5 ml disruption buffer (30 mM Tris-HCI (pH 7.4), 100 mM NaCl, 5 mM EDTA, 1% sodium dodecyl sulfate (SDS), 100 µg/ml proteinase K) . The cell suspension was disrupted by sonication for three 10-second intervals at 30 watts with the microtip of a Branson sonifier and incubated 60 min at 37°C . The lysate was extracted twice with phenol :chlorofrom (1 :1), once with chloroform/ isoamyl alcohol (24 :1) and precipitated with ethanol . The precipitated nucleic acids were redissolved in 20 mM Tris-HCI (pH 8), 10 mM MgCl 2 , 2 mM CaC12 , 100 µg/ml proteinase K-treated deoxyribonuclease I (Tullis and Rubin, 1980) . After incubation for 60 min at 37°C, SDS, EDTA, and NaCl were added to final concentrations of 1%, 50 mM and 0 .2 M, respectively, and the remaining nucleic acids (RNA) purified by phenol-chloroform extraction and ethanol precipitation . Preparation of Hybridization Probes Plasmid DNA was prepared essentially as described by Holmes and Quigley (1981) except that detergent was omitted . Plasmid DNA was further purified by two bandings in CsCI/ethidium bromide gradients . Restriction fragments to be used as hybridization probes were purified unlabeled by polyacrylamide gel electrophoresis and recovered by elecro-elution . Purified fragments were treated with 0 .5-1 U calf intestine alkaline phosphatase for 30 min at 37°C . The phosphatase was inactivated by heating for 15 min at 66°C and removed by phenol-chloroform extraction . The dephosphorylated fragments were recovered by ethanol precipitation . End-labeling reactions were carried out in 20 µl reaction mixtures containing 250 uCi -y-'P-ATP, (prepared according to Johnson and Walseth, 1979) and 5 U T4 polynucleotide kinase (PL Biochemicals) . Reaction conditions, recovery of labeled DNA, and strand separation procedures were as described by Maxam and Gilbert (1980) . S1 Nuclease Mapping End-labeled, single-stranded hybridization probes (10,000-50,000 cpm) were mixed with 20 µg in vivo RNA or with 20 µg carrier tRNA and precipitated with ethanol . The precipitated nucleic acids were washed with 70% ethanol, dried briefly under vacuum, and carefully redissolved in 10 µl of 40 mM PIPES (pH 6 .4) 0.4 M NaCl, 1 mM EDTA. Hybridization mixtures were incubated under paraffin oil for 5 min at 90°C and rapidly transferred to a 66°C water bath for 3 hr. Hybridization was terminated by dilution into 200 µl ice-cold S1 digestion buffer (30 mM sodium acetate [pH 4 .6], 0.25 M NaCl, 1 mM ZnSO 4, 5% glycerol, 20 µg/ml sonicated denatured salmon sperm DNA) containing 2000 U S1 nuclease (Boehringer-Mannheim; note that I U as defined by the manufacturer catalyzes the formation of 1 µg acid-soluble nucleotides in 30 min at 37°C) . After incubation for 30 min at 37°C, protected hybrids were recovered by ethanol precipitation, rinsed, and redissolved in loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0 .04% dyes) . Samples were denatured for 2 min at 90°C, quickchilled in ice-water, and analyzed by electophoresis in 8% polyacrylamide gels containing 8.3 M urea. After autoradiography, the portions of the gels which contained the protected fragments were excised and counted in a liquid scintillation counter . Other Methods In vitro transcription reactions were done as described previously (Gilman et al ., 1981) . RNA was purified from these reactions by digestion with proteinase K-treated deoxyribonuclease (Tullis and Rubin, 1980), phenolchloroform extraction, and ethanol precipitation . DNA sequencing reactions

Developmental and Genetic Regulation of Bacillus subtilis 293

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