(~) INSTITUTPASTEuR/ELSEVIER Paris 1991

R~'S. Microbiol. 1991, 142, 841-845

Expression of stage II genes during sporulation in Bacillus subtilis C . P . M o r a n Jr.

Department of Microbiology u,ld Immunology, Emery University School of Medicine, Atlanta, GA 30322 (USA) SUMMARY TWO RNA polymerase sigma factors, aF and ~E are produced dudng the first two horns of endospote formation in Bacillus subtilis, Transcription of the structural genes for these factors is activated about one hour after the start of andospore formation. The operon encoding v is transcribed by RNA polymerase containing ~H, another secondew sigma factor, whereas the operon encoding E is transcribed by RNA polymerasn containing a a, the primary sigma factor in growing sells. Evidently, the coordinate ternFeral control of these transcriptional units is med!ae.ed by n fact,or o;;ier than the sigma factors, possibly by the DNA-binding protein encoded b~¢spoDA. Both, r and ~e activities are also regulated by mechanisms operating after transcription. Key-words: Bacillus subtiiia, a Factor, spoOA Gone, Sporulafion. Transcription; Regu-

lation, Temporal control.

Introduction

AS the bacterium Bacillus stlbtilis differentiates from the vegetative form into a dormant endospore, complex morphological and physiological changes occur that require the expression o f over 50 genetic loci (for review, see Losiek and Krne~. !9...9; .~Ao ran, 1989). Several different sigma factors are produced during this process, replacing one another and conferring on the RNA polymerase different specificities for the recognition o f promoters. These ~eeondary sigma factors play a fundamental role in the regulation of sporulation gene expression. Two sigma factors, ~F and E , are produced during the first two hours after the onset of speculation. Both of these factors are required for development of the endospor¢ to proceed past stage 11, at which the asymmetric septum is completed, to stage I11, at which the fo~espore protoplast is engulfed by the mother cell protoplast. The exact role of these factots is unknown, but reguladoo of their synthesis and activity presumably plays a pivotal role leading to the commitment of the cell to differentiation, and in de-

termination of forespore and mother cell fates. In this report, 1 review recent results concerning the regulation of synthesis and activity of crF and r Regulation o f o F synthesis and aeti.Jity

The structural gene for ~r, spolIAC, is part of the spoHA operou, which consists of two additional sporulation genes, spoIlAA and spollAB (Fort and Piggot, 1984). Transcription of the spoIlA operon is activated about one hour after the onset of sporulation ( W u e t ~tl., 1989). The effects of integrated ialc,~mids, fusions of the spollA operon to lacZ, deletion analysis and primer extension axaalyses of transenpts hace been used to define thespollA promoter (We et eL, 1989, 1990). Transcription from the spollA promoter is completely dependent on the product of spoOH, wh.~ehiz ~.he secondary sigma factor oH ~'Wu eeaL, 1989). RNA polymerase containing qH w a s ~hown to use the spollA promoter in vitro (Wuet aL, 1990); therefore, it is likely ~hat H directs transcription of the spol[A operon in vice.

842

C.P. MORA N JR.

In addition to crH, other spoO gone products are required for transcription ofspol/A (Piggot and Curtis, 1987; Wu er aL, 1989). Evidently, the DNAbinding protein encoded by spoOA binds to sites upstream from the spollA promoter (J.A. Hoch, presented at the Bacillus Genome Conference, Paris 1990). Moreover, J. Hoch and his coworkers showed that the phosphorylatcd form of Spo0A can activate transcription from the spollA promoter by E~ H in vitro. Phosphorylation of Spo0A appcarz 1o be a prerequisite for the onset of sporulation (Perego et aL, 1989; Olmedo et al., 1990; Antoniewski et aL, 1990) and therefore spoHA transcription. Together, these results strongly favour a model in which phosphotylation of Spo0A leads directly to activation of the spollA promoter in rive. However, it is also known that the concentration of ~u increases about 4-fold during the transition from the exponential growth phase to stationary phase (Healy et aL, 1990). It is not known if this increase in H concentration contributes significantly to the activation of the spollA promoter. TranscriEtion o f the spollA operon is expected to lead 1o o ~ synthesis and therefore to the activation of promoters used by E= F. However, recent results snov, timt tie pioducts of spollAA and spollAB regulate the activity of s v (Sehimdt et aL, 1990). The product of spoHAB (SpolIAB) is a negative regulator of forespore-specifie gone expression (Rather et al., 1990). This effect of SpolIAB is due, at least in part, to its ability to antagonize ~v function (Sehimdt et aL, 1990). How SpolIAB antagonizes a ~ activity is not known. Three roles for SpoIIAB have been proposed (Rather er al., 1990; Schimdt et al., 1990). By stage 111 of endospOr¢ development, different sets of genes are being transcribed in the mother cell ar.d forespore (reviewed in Sallow, 1989; Losick and Kroos, i989). Since a '~ can direct the transcription of spolllG (Schimdt et al., 1990), which encodes the structural gone for the forespore-specifie sigma factor ~.G it is possible that SpoIIAB antagonizes synthesis o f a ~ in the mother cell compartment. In this way, SpolIAB may play a central role in determining cell-type-specific transcription. In addition to this role or as an alternative to this role, SpolIAB may act to delay oF-directed gene expression during sporulation. In !his role, SpolIAB welO,~ regulate the temporal expression of ~ -dependent genes. A third role for SpoIIAB is seen in cells that enter stationary phase in medium in which sporulafion does not occur. In these cells, the spollA operon is expressed, but the spollAB product prevents subsequent expression of forespore-speeific genes (Rather et aL, 1990). The spollAA product is necessary for or activity during sporaiation (Scbimdt el aL, 1990). SpolIAA may act by antagonizing SpollAB, since a mutation in sb,olIAB was found to bypass the requirement o f

SpolIAA for ~F-directed transcription during spnrulation (Schlmdt et aL, 1990]. It is unknown how SpolIAA interacts with or inhibits the activity o f SpollAB.

Regulation of ~z synthesis The structural gone for r_, siRE, is part of the spollO operon (Kenney and Moran, 1987). Deletion and complementation analysis demonstrated that siRE is essential for sporulation (Kenney and Moran, 1987). In addition to siRE, this operon includes a promoter proximal gene, spollGA, which is also essential for sporulation (Kenney and Moran, 1987). The product ofspoIIGA is required for the proteolyric processing o f the primary product o f siRE, pro-~ ~ (Jonas et at., 1988; Stragier et ul., 1988). Transcription of the spollG operon is activated about one hour after the onset of sporulation (Kennay and Moran, 1987 ; Kenney et al., 1988). We used integrating plasmids, promoter probe ptasmids, and primer extension analysis to determine the location of the spollG promoter (Kenney and Moran, 1987 ; Kenney et aL, t988). We al~o created transcriptional fusions of the spollG promoter and lacZ from E. co//(Kenney and Moran, 1987; Kenney et at., 1988). These fusions were inserted into the $P[~ phage chromosome and these phage used to lysogenize wildtype and mutant strains of B. subtilis. The minimum sequence of the spolIG promoter needed for full activity is shown in figure 1. The sequence of the spollG promoter shows that its - i0 region differs by only one base from the consensus sequence found at the - 1 0 regions of promoters used by a A RNA polymerase (Kenney et aL, 1988). Furthermore, a sequence found centred at position - 37 is identical to the sequence found at the - 35 regions of promoters used by ~A RNA polymerase. The unusual feature of this promoter is that the - 35-1ike sequence is separated from the - 10 sequence by 22 bp rather than the typi~zal 17 or 18 bp. It seems that the - 35-like sequence is about half a turn of the D N A helix too far upstream to function as a sigma contact site. Nevertheless, single base pair substitutions at these - 10- and - 35-like sequences in the spo11G promoter reduce activity of the promoter in rive (Kenney et al,, 1989) (fig. 1); therefore, these sequences play an essential role. We found that ~" R N A polymerase can use the spoHG promoter in vitro (Kenney et al., 1988). However, the best evidence that a A RNA polymerase uses this promoter in rive is our observation that a single amino acid substitution in a a suppressed the effect o f a single base pair substitution in the - 10 region of the spollC, promoter (Kenney et aL, 1989). Substitution of ar~inine for the glutaminc residue at position 196 of o suppressed the deleterious effect

STAGE H GENE EXPRESSION DURING B. SUBTILIS SPORULATIO1V

843 -400%

1

56G

C~B/t~.;I'R'CCCA£:acg~I,C.-,CTTC~'Tlri'ATAC'IffAT~

°

!Ill

-10%

7~

,116 -10(:

fig. I. The effects of single base pair substitutions in the ~pollGpromoter on expression of spollGlaeZ transcriptional fusions. The sequence shown is the non-transcribed strand and includes the minimal region required for wild-type promoter activity, "[he start point of transcription is indicated as + I. ~-gaiactosidaselevels were monitored at hourly intervals after the end of exponential growth. The arrows indicate the activity of each mutated promoter relative to the wild-type promoter, which is given the value of 100 %. The value5indicatedreflect the levelof [3-galaaosidaz¢2 h after the end.of exponentialgrowth.

of a transition at position -11 of the spolIG promoter. This suppression was allele-spgcJfie; i.e., the effects of other base pair substitutions at this or other positions in the spgHG promoter were not s u ~ pressed by this substitution in a Evidently, directly interacts with the - 10 region of the spollG promoter in vivo. In subsequen- experiments, we found that substitution of histidine for the arginine at ~.nsitioa 347 c.f ,~a specifically suppressed the effects of basc p~,J: substi(utiou~ a~ g.,'~.sitioi~- 34 of two pronao~eis t1,-~t are used by A during the exponential growth phase fires and iacBS, a derivative of the lac promoter from E. eoh) (Keuney and Moran, unpublished data), This substitution in a did not suppress the effects o f any o f the substitutions in the -35-like seaqttence of spollG. Evidently, the interaction of o with the sl;-ollG promoter during sporulation differs from its interaction with more typical promoters that are used during growth. We have examined the effects o f a large number o f single base pair substimtions in ihe spollG promoter (Satola et at., 1991) (fig. 1). These mutations were produced by oligonucleotide-directed mt,tagenesis and the effects assayed by constructing h Z fusions, which were carried on a SPas propbage.

The examination of the effects of mutations in the spollG promoter revealed three regions that are essential for promoter actwity: the - 1 0 region, a region centred around position - 3 7 and another centred around position - ~ 7 (fig. 1). The - 10 region sequence is important because of its intetactiocz with uA P.NA polymerase. Allelespecific suppression of the effect of a transition at ~,osition - 11 by an amino acid substitution in a p' provides strong evidence that c~a interacts with this region of the promoter (Ker,ney et al., 19891. Furthermore, the mutations in the - [0 ofspollG that changed this sequence so ~hat it was less similar to the canonical sequence that signals recognition by Eo a, reduced promoter activity. The one change in this region that increased ;}romoter activity changed the o-equence so that it resembled the canonical sequence perfectly (fig, I), The - 87 and - 37 regions may function as binding sites for one or more ancillary factors that regulate transcription from this promoter. Since we found that the product of spoOA is necessary for promoter activity in vivo (Kenney and. Moran, 1987) and Spo0A is a DNA-binding protein (Strauch et aL, 1990), we used ~el mobility shift assays and DNAse footprinting to examine binding of Spo0A to the

844

C.P. M O R A N JR.

spollG promoter (Satola et aL. 1991). DNase footprinting showed that Spo0A binds specifically to two regions of the spollG promoter, a high affinity site around - ~;7 and a low affinity site around - 37. The positions of the regions that are bound specifically by Spo0A in vitro correspond closely to the regions that were shown by the mutatinual analysis to be important for promoter activity. Because of this correlation, it is likely that the effects on promoter activity of the mutations in these regions can be explained by their effects on binding of Spo0A in vine. In fact. the - 87~, - 45G, and - 37C substitutions reduced binding of Spo0A to these sites in ritro in DNase footprinting assays (Satola and Moran. unpublished results). The - 3 5 C substitution increased the affinity of SpoOA for this site in vitro. We have not eliminated the possibility that factors other than Spo0A also interact directly with these regions of the promoter, but currently there is no evidence that an additional factor is involved. Since a mutation at position - 8 7 that reduces binding of Spo0A to ~hi: region i~: vftro a!so reduce,~ promoter activity in vine, it "~slikely that binding of Spo0A to this region is required for promoter activation. Most of the mutations in the region of the lower affinity Spo0A-binding site (the - 3 7 region) also reduce promoter activity; therefore, it is likely that interaction of Spo0A at this region is also required for aetivatioa of the promoter in vine. The one mutation in this region ( - 3 8 C ) that increases promoter activity increased binding of Spo0A to this site in vilro. The role of the high affinity Spo0A-binding site located at - 87 may be to increa-,e ~po0A binding to the low affinity slte at - 3 7 by n cooperative mechanism, but this has not been demonstrated. We have found that binning of Spo0A to the upstream site induced DNase[-hypersensltive sites located at ten bp intervals between the two Spo0A-binding sites. These could be caused by a bend in the DNA which cou!d facilitate the interaction o f SpOgA at the two sites (fig. 2). Furthermore, we found that a spoilO promoter with two base pair substitutions, - 87T and - 3 8 C , exhibked wild-type activity. It may be that increased binding of Sp00A to the - 37 region of the mutant r.r omelet with the - 87T an6 - 38C substitutions suppresses the requirement for a high affinity site at the upstream position. Since phosphory[ntion of Spo0A appears to signal the onset of sporulation (Olmedo et aL, 1990), we expect that the phcsphorylated form of Spo0A activates the spollG promoter. It E -or known whether phosphorylgtion of Spo0A increases its affinity for either of the two binding sites at the spolKi promoter or whether this phosphorylation affects the activity of the bound Spo0A. The promoters discussed in this review, spollA and spoHG, are acti,,ated during the first hour of sporulation ; hnweve., the spo11A promoter is used

-87

.....

~ii~!~!~

i!i

+~i~.......

Fig. 2. A model for Spo0A activation of the spollG promoter. This model illustrates that in order for RNA polymeruse to use the spollG promoter, ~' must interact with the - 10 region of the promoter and that SpoOA must interact at two sites, near positions -87 and -37. It is not known if SpoOA interacts with ~'. it is also not certain if or how SpoOA induces a bend in the DNA (see text).

by RNA polymerase containing n H (Wu et el., 1990) whereas, spollG is used by RNA polymeraSa containing ~r~' (Kenney et at., 1989). The activities of both o f these promoters is dependent on SpoOA. It may be .'hat the coordinate temporal control of these promoters is regulated by Spo0A and not by their cognate sigma factors. Acknowledgements

Work in the author's laboratory was ~t,pr~orted by Public Health Servicegrant A]20319, References

Antnniewski, C., gavelli, B . & Slr~ier, P. (1990), The spollJ gene, which regulatesearly developmentalsteps in Bacillus subHlis, belongs to a class of environmentally responsive genes. :. Beet,, 172, 86-93. Fen. P. & Piggot, P.J. (1984), Nucleotide sequence of sporuladon locus spoHA in Bacillus sublilis. J. gen. Microbiol., 130, 2147-2153. Healy, 3, Nair, G., Wdr, J., Smith, I. & Loslck, R. (lge~o), Pc:;t-tzanscriptim~at control of a sporulation regulatory geue eaeod;~l:, transer:.~tion fee or vH in Bacillus gubtili,+ i~foLMicrabiel. (in p~'esS). Jonas, R.M.. '~+'~-aver,E.A., Kenrtey, T.J., Moran, C.P. Jr. & Haldenwang, W.G. (1988), The Bacillus subtilis spollG oPeron encodes both oE and A gene necessary for