JOURNAL OF BACrERIOLOGY, June 1992, 0021-9193/92/113570-07$02.00/0

p.

Vol. 174, No. 11

3570-3576

Copyright © 1992, American Society for Microbiology

Roles of rpoD, spoIIF, spoIIJ, spoIIN, and sin in Regulation of Bacillus subtilis Stage II Sporulation-Specific Transcription PAM LOUIE, ANGELA LEE, KATHARINE STANSMORE, RICHARD GRANT, CHARLES GINTHER, AND TERRANCE LEIGHTON*

Department of Biochemistry and Molecular Biology, University of Califonia, Berkeley, Califonia 94720 Received 2 December 1991/Accepted 31 March 1992

A transcriptional switching process regulates the conversion between exponential and post-exponential-phase cell states in Bacillus subtilis (5, 10, 11, 23, 33). Under nutrientsufficient conditions cells remain in a vegetative mode. However, when nutrients, either singly or in combination, become limiting, growth slows, causing the organism to enter one of several possible post-exponential-phase states. It has been hypothesized that the nutrient-sensing mechanism is of the two-component sensor-transducer type (1, 5, 20, 21, 33). This model predicts that cells defective in either setisor or receiver molecules would be unable to activate post-exponential-phase functions, including sporulation. Several potential elements of the post-exponential-phase sensory chain have been identified in B. subtilis by analyzing the effects of second-site suppressors such as rvtA, sof, coi, and crsA (17, 19, 20, 28-30). Cells containing these intergenic suppressors can sporulate in the absence of a functional spoOB, spoOE, spoOF, or spoOK gene product. These spoO genes regulate the activation of spoOA, a phosphoryl receptor which is thought to be the master regulatory element controlling initiation of the sporulation process (1, 5, 10, 17, 19, 20, 21, 23, 25, 27, 29, 30). The rvtA, sof, and coi suppressor mutations map within the spoOA gene (15a, 20, 30). These intergenic suppressor mutations could produce SpoOA proteins that are more easily phosphorylated or less easily dephosphorylated or that can "cross-talk" more efficiently with noncognate kinases. The upstream sensory chain functions of spoOB, spoOE, and spoOF suggest that these genes are normally required to regulate and activate the spoOA gene product. The crsA mutation maps within the vegetative sigma factor (RpoD, &r') (26) and has been shown to be an intergenic suppressor of mutations in spoOB, spoOE, spoOF, and spoOK gene products (17). Recently, the effect of crsA and rvtA on strains containing various spoII to spoV mutations was determined (16). The crsA suppressor restored sporulation only in spoIIN, *

spoIIF, and spoIIJ strains. The genes suppressed by crsA, by analogy to the suppressible spoO mutations, became candidates for components of a sensory chain regulating the transition between sporulation stage 0 and stage II. The spoIIJ gene is known to encode a two-component regulatory system sensor element, kinA (1, 5, 21). The spolIF mutation has recently been characterized as a deletion spanning the 5' end of the kinA gene (18a). The spolIN mutation has been characterized as a missense mutation in the cell division regulatory gene ftsA (13). Further analysis of the spoIIN, spoIIF, and spoIIJ strains revealed that they are unable to transcribe spoIID, a late stage II gene (16). The mutated kinA and ftsA gene products could affect transcription of spoIID by (i) directly altering activity of the spoIID promoter or (ii) affecting the minor sigma factor c&', part of the transcription machinery necessary for recognizing the spoIID promoter (1, 31, 32). Mature sigma E is produced from the spoIIGB presigma primary translation product by the SpoIIGA protease (31, 32). The spoIIGA protein is postulated to remain inactive unless it is targeted to the asymmetric septal membrane (31). This targeting requires products of the spoIIA and spoIIE operons (12, 31, 32). We report here that in kinA and ftsA mutant backgrounds, the genes necessary for production of mature au are not efficiently transcribed. However, introduction of crsA into spolIN, spoIIF, and spoIllJ strains restores transcription of spoJIGAB, spollAC, and spollE operons to levels near or higher than those in wild-type strains. In addition to the spoO and spoII genes, there are several negative regulatory elements that are involved in sporulation regulation: hpr (22), abrB (24), and sin (8-10). The expression of at least one of these repressor genes, sin, is shown here to be inhibited by crsA. In the presence of high levels of sin, sporulation genes are not transcribed, and instead genes required in the search for new nutritional sources, such as those controlling motility and chemotaxis, are activated (10). Consistent with this interpretation of the role of sin is the finding reported here that sin deletions are intergenic suppressors of spolIN, spolIF, and spoIlJ mutations.

Corresponding author. 3570

Downloaded from http://jb.asm.org/ on January 20, 2019 by guest

Bacillus subtilis strains containing defects in the sporulation gene spoIIF (kinA), spoIU (kinA), or spoIIN gene spoIID. Results presented here and by other workers demonstrate that the spoIIF, spoIIJ, and spoIIN gene products control spoIID transcription indirectly by coordinating the induction of the spoIIGAB, spoIIE, and spoIJAC operons, which are required for crE synthesis and processing. Sporulation competence and spoIIGAB, spoIIE, and spoILAC transcription were restored in spoIIF, spoIJJ, and spoIIN mutants by introduction of crsA47, a mutation in the major vegetative sigma factor CA. crsA mutations are known to restore sporulation in certain spoO mutants. crsA suppression of kinA and ftsA mutations was achieved through inhibition of the transcription of sin, a gene involved in the selection between several post-exponential-phase cell states. A deletion of sin restored sporulation competence in spoIIF, spolIJ, or spoIIN mutant strains. A sin deletion was also able to restore sporulation competence in the crsA suppressible stage 0 mutant spoOK141.

(ftsA) cannot transcribe the cE-dependent

VOL. 174, 1992

B. SUBTILIS STAGE II SPORULATION-SPECIFIC TRANSCRIPTION REGULATION

3571

TABLE 1. B. subtilis strains Strain

"

Wild type 168 metC2 lys-I spoOF221 lys-] pheA1 crsA47strA spoIIF96 trpC2 spoIIF96 crsA47 spoIIJ::Tn917 trpC2 spoIIJ::Tn9J7 lys-J spoIIJ::Tn917 crsA47 spoIIN279 (Ts) metC3 rpoB2 spoIIN279(Ts) crsA47 metC3 spoIIN (spoIIE::lacZ) spoIIN crsA (spoIIE::lacZ) spoIIF96 (spoIIE::1acZ) spoIIF96 crsA47 (spoIIE::IacZ)

spoIIJ::Tn9J7 (spoIIE::lacZ) spoIIJ::Tn9J7 crsA (spoIIE::lacZ) spoIIN (spoHIGAB::lacZ) spoIIN crsA (spoIIGAB::lacZ) spoIIF96 (spoIIGAB::lacZ) spoIIF96 crsA47 (spoIIGAB::lacZ) spoIIJ::Tn917 (spoIIGAB::lacZ) spoIIJ::Tn9l7 crsA (spoIIGAB::lacZ) spolIN (spoIL4C::1acZ) spoIIN crsA (spoIIAC::lacZ) spoIIF96 (spoIIAC::lacZ) spoIIF96 crsA47 (spoIIAC::lacZ) spoIIJ: :Tn9J7 (spoIIAC: :lacZ) spoIIJ::Tn9J7 crsA (spoIIAC::lacZ) metB5 hisAI leuA8 (sin::lacZ Cmr) metB5 hisAI leuA8 (Asin Cmr) metB5 hisAl leuA8 (ORF-1::lacZ Cmr) 168 metC2 lys-I (sin::lacZ Cmr) 168 metC2 lys-] (ORF-1::lacZ Cmr) spoIIF96 Asin spoIIJ: :Tn9l 7Asin spoIIN279 Asin spoOK141 trpC2 spoOK141 crsA

These laboratories These laboratories 29 I. Takahashi BGSC 16 Alan Grossman AG522 td-168 met lys (met' selection) 15a BGSC 16 These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies These studies 8 9 8 These laboratories These laboratories These laboratories These laboratories

These laboratories BGSC These laboratories

BGSC, Bacillius Genetics Stock Center; td, gene transfer by transduction.

MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophage. The strains used, their genotypes, and their sources are listed in Table 1.

The crsA47 mutation was introduced into spoII lys-] strains by cotransduction with lys-] (17, 29). The recombinant suppressor phenotypes were confirmed by transductional backcrosses into RS4003 (spoOF221 lys-] pheA 1). The methods for construction of the spoIIGAB::1acZ, spoIIAC::lacZ, and spoIIE::lacZ fusion strains and transfer of the fusion to an integrating plasmid (pDH32) have been described elsewhere (1, 31). The sin::lacZ fusion, ORF-J::lacZ fusion, and sin deletion plasmids were provided by Issar Smith. The plasmids containing these constructs were transferred into various strains by selecting for the vector-associated chloramphenicol resistance gene. 13-Galactosidase synthesis by B. subtilis lacZ fusion strains. The expression of lacZ fusions was determined as described previously (7). Specific activity is expressed as nanomoles of o-nitrophenyl hydrolyzed per minute per milligram of cellular protein. Cell growth, induction of sporulation, and sporulation quantitation. Cell growth, induction of sporulation in Schaef-

fer's medium, and sporulation quantitation were performed as described elsewhere (29). RESULTS Effects of spoIIN, spoIIF, and spoIIU mutations with and without crsA on transcription of the spoIIGAB operon. The effects of spoIIN and spoIIF mutations on expression of a spoIIGAB::lacZ fusion are shown in Fig. 1 and 2. The transcription level of spoIIGAB::lacZ in a strain containing spoIIF was approximately 12% of the maximal wild-type level. Introduction of crsA into this strain restored transcription of spoIIGAB to levels elevated approximately 30% above wild-type levels. Similar results were obtained in spoIIJ mutant backgrounds (data not shown and reference 1). In the spoIIN(Ts) strain, expression of spoIIGAB::lacZ occurred at wild-type levels when the cells were grown at 35°C (the permissive temperature for the spoIIN mutation), although expression was delayed approximately 2 h. The crsA strain expressed spoIIGAB at twice the level expressed by an isogenic wild-type strain at 35'C. However, the spoIIN strain produced significantly decreased levels of spoIIGAB

Downloaded from http://jb.asm.org/ on January 20, 2019 by guest

168 RS1725 RS3002 Glu-47 IS59 RS3080 AG522 RS3150 RS3170 IS62 RS5061 RS6000 RS6001 RS6002 RS6003 RS6004 RS6005 RS6010 RS6011 RS6012 RS6013 RS6014 RS6015 RS6020 RS6021 RS6022 RS6023 RS6024 RS6025 IS424 IS432 IS423 RS6050 RS6060 RS6070 RS6080 RS6090 1S28 RS5054

Source or reference"

Genotype or phenotype

3572

J. BACTERIOL.

LOUIE ET AL.

20

spoI I5AB::IaCZ

50

a)

40

38.5C

o

_ >- I)

A

15

~0

0

4 U u L-II;

it. 30 _ 0)

LO

D

,

co %...-

10

\

5

10 ~

~

.

0 0 1

A

I

0 O

1

2 3 4 TIME (hrs)

5

6

5

6

40 >>

product at 40.5°C (nonpermissive temperature for spoIIN). The introduction of crsA into the spoIIN spoIIGAB::lacZ fusion strain restored spoIIGAB transcription to near-wildtype levels. Effects of spoIIN, spoIIF, and spoIIl mutations with and without crsA on transcription of the spoIJAC operon. Figures 3 and 4 present the effects of the crsA-suppressible genes on spoIL4C::lacZ transcription. In general, the effects seen were analogous to those observed in the case of spoIIGAB. The effect of spoIIF on the expression of spoIL4C was determined at 37°C. In the spoIIF strain, spoILAC expression was less than 10% of that observed in the isogenic wild-type strain. The spoIIF crsA strain produced wild-type levels of spoIIAC::lacZ product. crsA alone caused levels of spoIlAC transcription significantly higher than those observed in wild-type strains. Similar results were obtained in the spoIIJ mutant background (data not shown and reference 1). The spoIIN strain exhibited normal expression of spoIL4C, although at delayed times, at the nonpermissive temperature. Introduction of crsA into this strain led to significantly higher levels of spoIL4C expression occurring at times analogous to the production period of the wild-type strain at both permissive and nonpermissive temperatures. Again, the crsA mutation alone caused hyperexpression of the spoILAC operon. Effects of spoIIN, spoRIF, and spoIL mutations with and without csn4 on transcription of spoIIE genes. The pattern of spoIIE::lacZ transcription observed in the spoIIF and spolIF crsA strains (Fig. 5) followed the general pattern previously observed. At 37°C, little spoIIE product was produced by the spoIIF strain relative to the wild-type background. However, introduction of crsA elevated spoIIE levels to over twice the wild-type level. The only observations that were not consistent with the general pattern of low-level expression of spoIIGAB, spofL4C, and spoIIE in the suppressible spoII strain, with

5

50

6

FIG. 1. Effect of the spoIIF96 mutation, in the presence and absence of the second-site sporulation suppressor crsA47, on the expression of a spoIIGAB::lacZ fusion. ,-Galactosidase production in spoIIGAB::lacZ spoIIF96 crsA47 (0), spoIIGAB::lacZ wild-type 168 (a), and spoIIGAB::lacZ spoIIF96 (-) strains.

2 3 4 TIME (hrs)

)

CD

30

I._-

_uto u < cg

(

_

0

60h

(n >--o: H-

3

0

< A5

o

_->C3

x 2

M U X,

coLco 1-

0

B -l

2

3

Strain and relevant genotype

Spores/ml

37 37 37 37 37 46 46 37 37

RS1725 168 RS3150spoIIJ::Tn917 RS6080 spoIIJ::Tn9J7 Asin lS52spoIIF96 RS6070 spoIIF96 Asin 1S62 spoIIN279(Ts) RS6090 spoIIN279 Asin 1S28 spoOK141 trpC2 RS5054 spoOK141 Asin

9.7 x 107