Compartmentalization of Gene Expression during Sporulation of Bacillus subtilis Is Compromised in Mutants Blocked at Stage III of Sporulation

Mutations in the spoIIIA and spoIIIJ loci disrupt the compartmentalizationof gene expression during sporulation of Bacillus subtilis.The breakdown in compartmentalization is not the cause of theirbeing blocked in spore formation. Rather, it appears to be aconsequence of the engulfed prespore's being unstable.

INTRODUCTION

Top

Introduction

Cell differentiation is one of the fundamental processes inbiology. Central to it is the compartmentalization of gene expression,so that different genes are expressed in different cell types.Spore formation by Bacillus subtilis has become a paradigm forthe study of cell differentiation in prokaryotes. During sporulation,B. subtilis divides asymmetrically, resulting in two unequal-sizecells, the smaller prespore and the larger mother cell. Followingsporulation division, conventionally designated stage II, geneexpression becomes compartmentalized. Distinct RNA polymerasesigma factors become active, ${sigma}$ ^F in the prespore and ${sigma}$ ^E in themother cell (12). The mechanisms for compartmentalizing theactivity of these sigma factors are not fully understood. Followingdivision, the prespore is engulfed by the mother cell. The engulfedprespore (also called the forespore) develops into the maturespore. The mother cell is necessary for this process but ultimatelylyses. Completion of engulfment (stage III) is associated withactivation of other sigma factors, ${sigma}$ ^G in the prespore and ${sigma}$ ^K inthe mother cell. Mutants that are blocked at stage III do notactivate ${sigma}$ ^G or ${sigma}$ ^K (12).

A genetic test to analyze the compartmentalization of gene expressionduring sporulation was recently developed (7). The test is basedon the sacB/SacY transcriptional antitermination system (2).The SacY protein regulates transcription of sacB (encoding levansucrase)by antitermination. In the absence of SacY, transcription ofsacB terminates in the region 5' to the sacB structural gene.When SacY is present, it binds to the RAT site in the nascent5' region, prevents termination, and so allows transcriptionof the sacB structural gene (2). Thus, SacY needs to be presentduring transcription of sacB in order to obtain levansucraseor, in our case, ß-galactosidase from a sacB'-'lacZtranslational fusion. The SacY protein is ordinarily subjectto complex regulation, and we used a truncated derivative, SacY(1-55),that is free from this regulation (8). By placing sacB'-'lacZand sacY(1-55) under the control of promoters directed by thesame or different sigma factors, we were able to confirm thatthere was essentially complete compartmentalization of ${sigma}$ ^F and ${sigma}$ ^E activity during sporulation (7).

We have used mutagenesis with a Tn10 derivative (9) to generatemutants with the compartmentalization of the activities of ${sigma}$ ^Fand ${sigma}$ ^E disrupted and have obtained mutations in the spoIIIA andspoIIIJ loci. Here we explore the reason why such mutants mighthave compartmentalization disrupted. We distinguished two alternativesthat would point to radically different roles for these spoIIIloci. The first is that the mutations disrupt compartmentalization,and as a consequence sporulation cannot proceed beyond stageIII. The second is that the mutations cause a block at stageIII, and as a consequence the engulfed prespores are prone tolyse, causing loss of compartmentalization. In the first mechanism,disruption of compartmentalization should be apparent as soonas ${sigma}$ ^F and ${sigma}$ ^E become active. In the second mechanism, the activitiesof ${sigma}$ ^F and ${sigma}$ ^E should be compartmentalized when they first appear,and compartmentalization should be lost only following engulfment.We found the latter to be the case, as the loss of compartmentalizationfollows the completion of engulfment.

Isolation of mutants disrupted in compartmentalization of gene expression.

Top

Isolation of mutants disrupted...

A pool of B. subtilis mutants with mini-Tn10 insertions (9)was screened for compartmentalization of ${sigma}$ ^F and ${sigma}$ ^E activities.The mutants were obtained in strain SL7643, which is a derivativeof SA501 cured of SPß (7); SA501 (8) has deletionsof sacY, sacB, licT, and sacT to avoid possible interferencewith the antitermination system being used. Chromosomal DNAof the pooled mutants was purified and used to transform thetester strain SL9106, also a derivative of SA501, selectingfor Tn10-associated chloramphenicol resistance. Strain SL9106contains P_cotEp1-sacB'-'lacZ/P_spoIIQ-sacY(1-55) (the ${sigma}$ ^E/ ${sigma}$ ^F configuration)(7). Among approximately 30,000 Cm^r transformants, 32 displayedß-galactosidase activity. Of these, one activatedthe cryptic endogenous lacA gene, and one mapped in spoIIIE,where insertions have previously been shown to disrupt compartmentalization(7, 17). The remaining Tn10 mutations were tested by backcrossinginto strain SL9106 and by transfer to strain SL9105 (which hasthe opposite configuration, ${sigma}$ ^F/ ${sigma}$ ^E). Nine mutations that causedsubstantial ß-galactosidase expression in both strainswere then transferred into strains with ${sigma}$ ^E- and ${sigma}$ ^F-directed gfpfusions (SL9109 spoIID-gfp and SL10280 spoIIQ-gfp, respectively[kindly provided by David Hilbert and Vasant Chary]). Eightof the mutations caused a substantial loss of compartmentalization,as visualized with bacteria grown on Schaeffer's sporulationagar (11). This result indicated that the sacB/SacY screen wasan effective predictor of loss of compartmentalization.

Genetic crosses between the eight strains with impaired compartmentalizationindicated that the mutations mapped in two distinct loci. FlankingDNA from the inserts was recovered and sequenced, and the lociwere identified as spoIIIJ (seven mutations) and spoIIIAB (onemutation; Tn10 insertion 428 nucleotides downstream from thepresumed start of translation). The ninth mutation, which resultedin ß-galactosidase activity in SL9105 and SL9106 buthad little effect on the compartmentalization of ${sigma}$ ^E- and ${sigma}$ ^F-directedgfp expression, mapped in ybdB (Tn10 insertion 457 nucleotidesdownstream from the presumed start of translation); it was notstudied further. The ybdB locus, now designated skf, is associatedwith production of an antibiotic that kills nonsporulating bacteriain the same population (4). The spoIIIAB mutation and a spoIIIJmutation with Tn10 inserted 472 nucleotides downstream fromthe presumed start of translation were used for further study.Mutations in the spoIIIJ and spoIIIA loci block sporulationat stage III and prevent activation of ${sigma}$ ^G and hence ${sigma}$ ^K (3, 10,12, 14, 15); the engulfed prespores of spoIIIA mutants havebeen reported to be unstable (10, 16).

Compartmentalization of gene expression is gradually lost during sporulation of spoIIIA and spoIIIJ mutants.

Top

Compartmentalization of gene...

The following questions arise for spoIIIA and spoIIIJ: do themutations directly affect the compartmentalization of ${sigma}$ ^F and ${sigma}$ ^E activity, or do they indirectly influence the process, becausethey block sporulation at the stage of engulfment, and the mutantprespore is unstable and breaks down, resulting in loss of compartmentalization?To distinguish between these possibilities, compartmentalizationof gene expression during sporulation was monitored by observingexpression of spoIIQ-gfp (directed by ${sigma}$ ^F) and spoIID-gfp (directedby ${sigma}$ ^E). We reasoned that if the primary effect of spoIIIA andspoIIIJ mutations is on compartmentalization, then we shouldbe able to observe impaired compartmentalization at the earliesttimes of green fluorescent protein (GFP) expression and thatthe extent of impairment should not increase with time duringsporulation. In contrast, if loss of compartmentalization wasa consequence of an engulfment or other defect, then GFP expressionshould initially be compartmentalized; bacteria displaying uncompartmentalizedexpression should appear only after they reach the impairedstage. It is thought that ${sigma}$ ^F and ${sigma}$ ^E continue to be active in theabsence of ${sigma}$ ^G and ${sigma}$ ^K activity (7), so that either mechanism forthe breakdown in compartmentalization would result in ß-galactosidaseactivity with the sacB/SacY system. The sacB/SacY system wasused to analyze populations and not single cells and so didnot satisfactorily distinguish between the two possibilities;expression of gfp was found to be more suitable for analyzingsingle cells.

Strains with spoIIQ-gfp and spoIID-gfp transcriptional fusionswere induced to sporulate in modified Schaeffer's sporulationmedium (11). They were examined by fluorescence microscopy atvarious times after the start of spore formation, defined asthe end of exponential growth. Conditions for microscopy wereessentially as previously described (5, 13, 18). When GFP fluorescencewas first detected, about 3 h after the end of exponential growth,it was almost completely compartmentalized in the spoIIIA andspoIIIJ mutants as well as the spo⁺ strain. This observationapplies to both ${sigma}$ ^F-directed (spoIIQ-gfp) and ${sigma}$ ^E-directed (spoIID-gfp)fusions. Expression remained largely compartmentalized for severalhours (Table 1). However, by 8 to 10 h after the start of sporulation,fluorescence became uncompartmentalized in an increasing proportionof the mutant bacteria (Table 1). The transition from compartmentalizedto uncompartmentalized expression was observed consistentlyfor the spoIIIAB and spoIIIJ mutants, although the extent ofthe breakdown in compartmentalized expression varied from experimentto experiment (data not shown).

View this table:
[in this window]
[in a new window]
TABLE 1. Effect of mutations in spoIIIAB and spoIIIJ on the compartmentalization of ${sigma}$ ^F- and ${sigma}$ ^E-directed gfp expression

In the sporulation system used, there is a substantial period,about 3 h, between the end of exponential growth and the formationof the sporulation septum, and by 5 h after the end of exponentialgrowth, 86 to 100% of the GFP-expressing population were atstage II of sporulation. Cultures were examined in detail 7h after the end of exponential growth, before there was significantbreakdown in compartmentalization. Engulfment was complete inabout 50% of the GFP-expressing bacteria of the spoIIIJ andspo⁺ strains and in a substantial portion of the spoIIIAB strain(Table 2) (in other experiments, that portion also reached 50%).Similar results were obtained with spoIIQ-gfp ( ${sigma}$ ^F-directed) andspoIID-gfp ( ${sigma}$ ^E-directed) fusions. Thus, completion of engulfmentoccurred well before substantial numbers of mutant bacteriadisplayed uncompartmentalized expression. It is inferred thatthe effect of the spoIIIAB and spoIIIJ mutations on compartmentalizationwas most likely a consequence of the instability of the engulfedprespore that led to mixing of mother cell and prespore cytoplasms.This effect contrasts with the loss of compartmentalizationcaused by insertional inactivation of spoIIIE, where the defectoccurs at stage II and is most likely the consequence of a smallpore in the septum permitting small proteins such as sigma factorsand GFP to cross the septum (17; D. Hilbert, V. Chary, and P.Piggot, unpublished observations). Inactivation of the spoIIABlocus results in uncompartmentalized ${sigma}$ ^F activity and blocks sporeseptum formation (1); the mutants display little ${sigma}$ ^E activityand so would not be detected with the sacB/SacY system. Theonly mutations causing severe defects in compartmentalizationthat we detected in 30,000 Tn10 mutants screened, with a systemrequiring both ${sigma}$ ^F and ${sigma}$ ^E activity, were located in spoIIIE (7),spoIIIA, and spoIIIJ. The transposon insertion could have apolar effect on genes downstream in the spoIIIA and spoIIIJoperons. We have not tested for this possibility. Disruptionof the gene downstream of spoIIIJ does not affect spore formation(3, 12), so that spoIIIJ inactivation is the most likely causeof the effect described here. Nonpolar mutations in each ofthe eight genes in the spoIIIA operon result in the same stageIII sporulation phenotype (12, 15), suggesting that disruptionof other genes in the operon would have the effect describedhere. Our results suggest that insertional inactivation of avery limited number of loci results in uncompartmentalized activitiesof both ${sigma}$ ^F and ${sigma}$ ^E.

View this table:
[in this window]
[in a new window]
TABLE 2. Stage of sporulation reached by bacteria displaying compartmentalized expression of ${sigma}$ ^F- and ${sigma}$ ^E-directed gfp fusions 7 h after the end of exponential growth

ACKNOWLEDGMENTS

We thank Bill Schenck for technical assistance.

This work was supported by Public Health Service grant GM43577from the National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Phone: (215) 707-7927. Fax: (215) 707-7788. E-mail: piggotp{at}temple.edu.

Present address: Fox Chase Cancer Center, Philadelphia, PA 19111.

REFERENCES

Top