The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent...

The Bacillus SpoIIGA protein is targeted to sites ofspore septum formation in a SpoIIE-independentmanner

Paul Fawcett, 1 Alexandre Melnikov 2 and PhilipYoungman 2*1University of Georgia, Department of Genetics, Athens,GA 30602, USA.2Millennium Pharmaceuticals Inc., Cambridge, MA02139-4815, USA.

Summary

The process of bacterial cell division involves theassembly of a complex of proteins at the site of septa-tion that probably provides both the structural and thecytokinetic functions required for elaboration and clo-sure of the septal annulus. During sporulation in Bacil-lus subtilis , this complex of proteins is modified bythe inclusion of a sporulation-specific protein, SpoIIE,which plays a direct role in gene regulation and alsohas a genetically separable role in determining thegross structural properties of the specialized sporula-tion septum. We demonstrate by both green fluorescentprotein (GFP) fusions and indirect immunofluores-cence microscopy that SpoIIGA, a protein requiredfor proteolytic cleavage of pro- sE, is also targeted tothe sporulation septum. Septal localization of SpoIIGA–GFP occurred even in the structurally abnormal sep-tum formed by a SpoIIE null mutant. We also reportthe isolation of a spoIIGA homologue from Bacillusmegaterium , a species in which the cells are signi-ficantly larger than those of B. subtilis . We haveexploited the physical dimensions of the B. megater-ium sporangium, in conjunction with wide-field decon-volution microscopy, to construct three-dimensionalprojections of sporulating cells. These projectionsindicate that SpoIIGA–GFP is initially localized in anannulus at the septal periphery and is only later local-ized uniformly throughout the septa. Localization wasalso detected in a B. subtilis spo0H null strain thatfails to construct a spore septum. We propose thatSpoIIGA is sequestered in the septum by an inter-action with components of the septation machinery

and that this interaction begins before the construc-tion of the asymmetric septum.

Introduction

A crucial step in endospore formation in Bacillus species isthe construction of a sporulation septum at an asymmetricposition proximal to a pole of the cell body. This polar sep-tation event effectively partitions the sporangium into twounequally sized compartments with distinct developmentalfates; the smaller prespore compartment develops into amature spore, while the mother cell eventually lyses andreleases the spore. These morphological changes are co-ordinated with changes in gene expression directed byRNA polymerase sigma factors that are activated in a com-partment-specific manner (for reviews, see Errington, 1993;Stragier and Losick, 1996). Inactive forms of both sF, thefirst prespore-specific sigma factor, and sE, the firstmother cell-specific sigma factor, are present in the cellbefore the synthesis of the polar septum (Gholamhosei-nian and Piggot, 1989; Lewis et al., 1996). The mechan-isms by which they become active differ but, in eachcase, compartment-specific activation requires a functionattributable to sporulation proteins targeted to the asym-metric septum. The activation of sF requires a functionalSpoIIE protein (Margolis et al., 1991), the septal localiza-tion of which has been previously reported (Arigoni et al.,1995; Barak et al., 1996; Levin et al., 1997), whereasthe activation of sE requires SpoIIGA (Jonas et al.,1988; Peters and Haldenwang, 1994), the septal localiza-tion of which we now report.

The formation of a cell division septum in eubacteria isbelieved to involve the concerted action of a complexassemblage of proteins that has been termed the ‘septator’or ‘septasome’ (Vincente and Errington, 1996). The mostextensively characterized septasome component is thecell division protein FtsZ. FtsZ is widely conserved through-out the bacterial kingdom and appears to be essential forviability in most species, including B. subtilis (Beall et al.,1988; Dai and Lutkenhaus, 1991). FtsZ undergoes in vitroself-assembly into structural homologues of tubulin poly-mers and has slight sequence homologies to tubulin(Bramhill and Thompson, 1994). During the Escherichiacoli division cycle, cytoplasmic FtsZ monomers aggregatein a circumferential ring at future division sites. This ring is

Molecular Microbiology (1998) 28(5), 931–943

Q 1998 Blackwell Science Ltd

Received 7 January, 1998; revised 27 February, 1998; accepted 2March, 1998. *For correspondence. E-mail [email protected];Tel. (617) 761 6816; Fax (617) 374 9379.

progressively constricted and remains at the leading edgeof the invagination of membrane and cell wall duringcytokinesis, leading Bi and Lutkenhaus (1991) to proposethat FtsZ performs a cytoskeletal function. The finding thatFtsZ interacts directly with other important division pro-teins, such as FtsA (Ma et al., 1996; Wang et al., 1997)and ZipA (Hale and de Boer, 1997), and appears to co-localize with others, such as FtsI (J. Pogliano et al.,1997) and DivIB (Harry and Wake, 1997), has led to thesuggestion that FtsZ may also act as a scaffold uponwhich the multiprotein septasome complex is built (Addi-nall et al., 1996).

The sporulation septum of endospore formers differsfrom the vegetative septum both in its site of assemblyand in its physical characteristics. Like the vegetative divi-sion septum, the sporulation septum is initially filled withcell wall material. However, the quantity of murein depos-ited between the lipid bilayers of the sporulation septum ismuch reduced, resulting in a visibly thinner structure. Inthe normal course of sporulation, all detectable murein isremoved from the polar septum to produce a pliable doublemembrane (Holt et al., 1975). This structure does notinvaginate to form separate division products, but rathermigrates toward the spore pole, eventually completelyengulfing the forespore compartment. Despite these dif-ferences, it is clear that many of the basic componentsof the assembly machinery are the same in both septumtypes. Thus, it has been proposed that the sporulation sep-tum should properly be viewed as a modified, specializedversion of the cell division septum (Hitchins and Slepecky,1969). How is the septasome scaffold modified duringsporulation, and how is the special septal structure thatforms during sporulation diverted to participate directlyin establishing compartment-specific programmes of geneexpression? The 92 kDa SpoIIE protein probably plays acentral role. Biochemically, SpoIIE is a serine protein phos-phatase and, in this capacity, it is directly responsible formodifying the interaction between SpoIIAA and SpoIIAB,which controls the activation of sF in the forespore (Dun-can and Losick, 1993; Diederich et al., 1994; Duncan etal., 1995; 1996; Arigoni et al., 1996; Feucht et al., 1996).SpoIIE is localized at sites of septation (Arigoni et al.,1995; Barak et al., 1996) and is also required for the con-struction of morphologically normal polar septa; spoIIEnull strains produce septa exhibiting a ‘straight and thick’phenotype typically associated with murein-rich vege-tative septa (Piggot, 1973; Illing and Errington, 1991).In addition, SpoIIE is required for the efficient initiationof polar septation, and this role in septum initiation andphenotypic determination is distinct and separable fromits role in sF activation (Barak and Youngman, 1996;Feucht et al., 1996). Because mutations at no othergenetic locus result in the thick polar septum phenotype,it has been suggested that SpoIIE could play a critical

role in modifying the nature of the septum-synthesizingscaffold during sporulation (Barak et al., 1996). For exam-ple, it might be supposed that spoIIE null mutants wouldfail to recruit other sporulation-specific components ofthe septasome.

Mother cell gene expression is initially directed by themodified gene product of spoIIGB. The spoIIGB locusencodes a sigma factor precursor, pro-sE, that is activatedby the proteolytic cleavage of 27 amino-terminal residues(Stragier et al., 1984; Trempy et al., 1985; LaBell et al.,1987). Genetic evidence indicates that the activating pro-tease is encoded by spoIIGA, a gene located upstream inthe same operon (Kenney and Moran, 1987; Stragier etal., 1988; Peters and Haldenwang, 1994). SpoIIGA hasslight homology to a family of aspartic proteases and ispredicted to be an integral membrane protein (Stragier etal., 1988; Peters and Haldenwang, 1991). The timing ofSpoIIGA-mediated pro-sE cleavage is controlled by anintercellular signalling protein, SpoIIR, which is producedin the forespore under sF control and is secreted acrossthe septum (Hofmeister et al., 1995; Karow et al., 1995;Londono-Vallejo and Stragier, 1995). Topological analysisof SpoIIGA based solely on the B. subtilis sequencesuggests a model with five amino-terminal membrane-spanning segments and a cytoplasmic carboxyl-terminalglobular domain (Stragier et al., 1988). These observa-tions make it attractive to suppose, as proposed by Stragier,that SpoIIGA is targeted to the sporulation septum andthat obligatory targeting to the septum serves as a mech-anism to co-ordinate morphogenesis with key changes ingene expression. To determine the subcellular localizationof SpoIIGA, we have used fusions of SpoIIGA to anenhanced intensity variant of the green fluorescent protein(GFP) of Aequorea victoria, and here provide evidencethat SpoIIGA from both B. subtilis and B. megaterium istargeted specifically to the sporulation septum. Moreover,we show that this localization is not dependent on the pre-sence of functional SpoIIE. Previous studies have beenunable to determine if localization of a protein at thesites of septation implies localization throughout the sep-tum or only to an annulus at the periphery of the cell. Toresolve this issue, we used immunofluorescent microscopy(IFM) in conjunction with wide-field deconvolution micro-scopy to create three-dimensional projections of sporulatingB. megaterium cells. These computerized reconstructionscan be rotated arbitrarily, allowing us to examine projec-tions down the long axis of cells. Using this approach,we find that both SpoIIGA and SpoIIE are initially concen-trated in an annulus corresponding to a site of septuminitiation, but that both eventually spread throughout theseptum. This is consistent with our observation that alow level of septal SpoIIGA localization can be observedin spo0H mutant strains in which sporulation is blockedbefore the construction of a polar septum.

Q 1998 Blackwell Science Ltd, Molecular Microbiology, 28, 931–943

932 P. Fawcett, A. Melnikov and P. Youngman

Results and discussion

Localization of SpoIIGA–GFP in B. subtilis

To determine the subcellular localization of SpoIIGA, weconstructed a temperature-sensitive integrational plasmid,pIIGA-GFP, which mediates chromosomal SpoIIGA–GFPtranslational fusions. Integrations of this plasmid result in aspoIIGA–GFP translational fusion under the control of thenatural spoIIGA promoter, accompanied by a separation ofthe downstream spoIIGB gene from its promoter. Chromo-somal integrations were made initially in a wild-type B.subtilis background, which established that SpoIIGA is tar-geted to sites of polar septum synthesis (strain PMF 9, Fig.1A). When grown in liquid sporulation medium at 308C,cells began to acquire a diffuse green fluorescence begin-ning approximately 1 h after the cessation of logarithmicgrowth (T1). The fluorescent signal slowly increased and,by T2, many cells exhibited localization at one or both cellpoles (Table 1). This localization presumably correspondsto sites of septum synthesis and is stable for at least 8 h.Because the integrative plasmid mediating the SpoIIGA–GFP fusion separates spoIIGB from the spoIIG promoter,PMF 9 was abortively disporic. Apart from the 53% of PMF9 cells that had localized signal at one or both cell poles,29% of cells never developed more than a diffuse glow,while the remaining 18% developed a signal that wasnot associated with the cell poles (Table 1). This lattertype of signal usually appeared as disorganized granularpatches; examination of such cells by phase-contrastmicroscopy often revealed damage or lysis. We attributethe relatively low percentage of cells that developed alocalized signal to the weakness of the GFP signal, reflect-ing the relative weakness of the spoIIG promoter. A slightalteration in the plane of focus often made a localizedsignal appear at one pole of the cell, while causing the sig-nal at the other pole to diminish or disappear. Use of a100 × objective lens in place of a 60 × lens also caused adecrease in apparent localized signal because of thereduced light-gathering ability of the instrument. Carewas therefore taken to maximize the strength of GFP fluor-escence (see Experimental procedures).

To test the hypothesis that SpoIIGA localization mightrequire a functional SpoIIE protein, we constructedspoIIGA–GFP fusions in spoIIE64 (PMF 10, Fig. 1B) andspoIIE ::Tn917VHU7 (PMF 14, Fig. 1C) genetic back-grounds. As a consequence of a missense mutation leadingto a defect in phosphatase activity, spoIIE64 strains aredefective in the expression of sF-dependent genes, butnevertheless produce polar septa of normal appearance(Barak and Youngman, 1996). The spoIIE ::Tn917VHU7mutation is caused by a transposon insertion in spoIIEnear the beginning of its coding sequence, and this pro-duces a true null phenotype characterized by thick septa

and a decreased frequency of septum initiation (Sandmanet al., 1987; Barak and Youngman, 1996). Polar localiza-tion of SpoIIGA–GFP fusion proteins occurred in all threegenetic backgrounds (Fig. 1A–C). We therefore concludethat SpoIIE plays neither a direct nor an indirect role in tar-geting SpoIIGA to the sporulation septum. An interestingpossibility was raised by the extent of localization observedfor PMF 14, the spoIIE null strain. While the total percen-tage of cells showing localization (classes A and B inTable 1) fell from 53% in PMF 9 to only 34% in PMF 14,the 34% of cells with localized signal in PMF 14 was greaterthan expected. While spoIIE null strains incubated forextended periods of time eventually septate at a frequencyapproaching that of other stage II mutants (I. Barak, per-sonal communication), there is clearly a significant delayin the onset of septation. This delay was documented inthe study of Feucht et al. (1996), which used phase-con-trast microscopy to follow the kinetics of septum formationin a spoIIE ::ermC null mutant strain. This study establishedthat only approximately 5% of spoIIE null mutant cellsexhibited a disporic phenotype at T2.5. A similar delaywas also reported by Barak and Youngman (1996), whoused the direct enumeration of septation frequenciesfrom electron micrographs to show that only approximately5% of spoIIE ::Tn917VHU7 cells harvested at T10 exhib-ited class A localization. In contrast, we observed classA SpoIIGA localization in 21% of PMF 14 cells harvestedat T2.5. We interpret this as consistent with the possibilitythat SpoIIGA–GFP is initially targeted to a preseptationalcomponent of the septasome rather than to the completedseptum structure. We speculate that SpoIIGA may be inter-acting with a protein or complex associated with the septa-some constructed shortly after the shift of the FtsZ ringfrom the mid-cell site to the site of asymmetrical division.The persistence of septal murein in spoIIE strains clearlyargues against models requiring SpoIIGA recognition ofthe thin double-membrane configuration that results fromthe removal of murein. The septal localization of SpoIIGAin spoIIE backgrounds also implies that localization doesnot require sF- or sE-dependent transcription. Whilecomplementation studies have shown that the SpoIIGA–GFP fusion protein is inactive (H. Peters, personal com-munication), we do not believe that this influences local-ization; it seems improbable that the disruption of normalSpoIIGA targeting would be accompanied by a gain offunction resulting in the precise localization of SpoIIGA–GFP at the highly specialized sporulation septum.SpoIIGA has a predicted membrane-spanning topology,and pro-sE, its proteolysis substrate, has been previouslyshown to localize at the sporulation septum (Stragier etal., 1988; Ju et al., 1997). It is therefore consistent topostulate that native SpoIIGA is also targeted to thepolar septum.


Localization of Bacillus SpoIIGA proteins 933

Localization of SpoIIGA in a B. subtilis spo0H mutant

To clarify whether SpoIIGA localization occurs in predivi-sional sporangia, we constructed a SpoIIGA–GFP fusionstrain in an a spo0H genetic background. The spo0Hlocus encodes a sigma factor, sH, that is most strongly

expressed during late log phase growth and is involvedin the transition to stationary phase (Weir et al., 1991).Upon entry to sporulation, spo0H null mutants undergoaxial filament elongation, but are blocked before theconstruction of the sporulation septum. Our choice of aspo0H mutant to abolish septation during this experiment


Fig. 1. Fluorescent and immunofluorescent microscopy.A–C. Localization of SpoIIGA–GFP-associated fluorescence at sites of polar septation in B. subtilis from culture samples harvested 2.5 h aftercessation of logarithmic growth (T2.5) The scale bar in (A) represents 3 mm and applies to A–C.A. PMF 9 (spoIIGA–GFP ), wild-type background.B. PMF 10 (spoIIGA–GFP, spoIIE64 ), a spoIIE missense mutant background.C. PMF 14 (spoIIGA–GFP spoIIE ::Tn917VHU7 ), a spoIIE null background.D–F. Cells of B. megaterium PMF 50 (spoIIGA–GFP ) from T2 and stained for IFM using an anti-GFP primary antibody and an Oregon Green488-conjugated secondary antibody. The scale bar in (D) represents 3 mm and applies to panels D–H.D. Both FITC and DAPI wavelengths shown.E. FITC wavelength only.F. DAPI wavelength only (nucleoids have been artificially coloured red).G. Computer-reconstructed projection down the long axis of a B. megaterium PMF 50 cell at T1.5, showing the early annular pattern ofSpoIIGA localization.H. Projection down the long axis of a PMF 50 cell from T5, showing disc-like localization.I. B. megaterium PY1272 cells (spoIIE–GFP ) stained for IFM as described for D–F. In (I), the lefthand cell has the straight septum typical ofearly points, while the righthand cell shows the SpoIIE cage, which forms around the forespore nucleoid. The scale bar in (I) is 3 mm andrefers to I–K.J. SpoIIE cages forming around the prespore of PY1272 cells as shown by GFP fluorescence alone.K. A composite micrograph of B. subtilis PMF 37 (spoIIGA–GFP, spo0H ::kan) cells. In (K), the top right cell shows normal localization (11%of cells), while the two bottom cells show the aberrant semi-circular pattern of localization seen only in this strain (approximately 5% of cells).


was influenced by the observation that the position of theFtsZ ring is correctly shifted from medial to polar positionsin such mutants (Levin and Losick, 1996). The FtsZ ring isknown to be essential for the localization of SpoIIE (Levinet al., 1997), and we considered it likely that predivisionalSpoIIGA localization would also require this fundamen-tal septasome component. This is in contrast to spo0Amutants, which fail to shift the FtsZ ring to the poles(Levin and Losick, 1996). The difficulty with this approachis that expression from the spoIIG promoter is significantlydiminished in spo0H strains (Kenney and Moran, 1987).Localization of SpoIIGA–GFP was difficult to detect inthe wild-type background and could not be detected in aspo0H mutant. To overcome this problem, it was necessaryto construct an integrative spoIIGA–GFP plasmid contain-ing the entire coding region of spoIIGA, as well as anupstream region required for maximal expression fromthis promoter. By training strains with chromosomal inte-grants of this plasmid on successively higher levels of chlor-amphenicol (up to a final concentration of 80 mg ml¹1), wecreated a strain containing multiple tandem duplications ofthe spoIIGA–GFP translational fusion in a spo0H ::kanbackground (Janniere et al., 1985; Piggot and Curtis,1987). These tandem repeats apparently partially com-pensated for reduced expression from the spoIIG promoter,allowing polar localization of SpoIIGA–GFP to be observedin this strain (Fig. 1K, cell at top righthand corner), albeit ata reduced frequency. We found that 11% (total n ¼ 234) ofthe slightly elongated spo0H filaments contained one ormore bands appearing at presumptive polar septationsites. We also observed that 21% of the filaments con-tained disorganized patterns of localization or normal local-ization together with disorganized patterns. As with thewild-type SpoIIGA–GFP strain, many of the cells with dis-organized localization appeared to be damaged or lysedwhen examined by phase-contrast microscopy. However,we reproducibly observed one unusual pattern not seen inany of the other strains in this study: approximately 5% ofthe cells showed a semicircular pattern of localization (bot-tom two cells in Fig. 1K). We speculate that this structure

might correspond to localization of SpoIIGA to incompleteor deformed septasomes that have formed at an obliqueangle relative to the long axis of the cell. The supplemen-tary ftsAZ promoter P2 is sH dependent, and the inductionof this promoter at T0 may be required for the efficientproduction of normal septation sites (Gholamhoseinianet al., 1992; Gonzy-Treboul et al., 1992). This localizationpattern may also be a consequence of reduced levels ofSpo0A or some other sH-dependent factor. This pheno-type is in some ways reminiscent of the occasional obliqueseptation event that has been reported during minicellformation in divIVA mutants (Edwards and Errington,1997).

Conserved features of SpoIIGA from B. subtilis,B. megaterium, Bacillus thuringiensis and Clostridiumacetobutylicum

To achieve enhanced resolution of SpoIIGA localization,we characterized the SpoIIGA homologue from B. mega-terium, a species in which the cell diameter is approxi-mately two times greater than in B. subtilis cells. Thishomologue was successfully isolated using a degenerateprimer/inverse polymerase chain reaction (PCR)-basedapproach and sequenced (GenBank accession numberAF0171810; see Experimental procedures). This approachassumed a preservation of the gene order found in B.subtilis, and we indeed found that the organization of theB. megaterium genome near the spoIIG locus is similarto that of B. subtilis (gene order: bpr – spoIIGA –spoIIGB ). A search of the GenBank database revealedthat in addition to the spoIIGA homologue of B. subtilis(Stragier et al., 1988; Masuda et al., 1990), those of C.acetobutylicum (Sauer et al., 1994) and B. thuringiensis(Adams et al., 1991) have also been cloned andsequenced (although only the carboxyl-terminus of the B.thuringiensis sequence is available). These sequenceswere aligned using the PILEUP module of the GCG suite(Devereux et al., 1984) to determine which regions ofthis protein are conserved and are therefore most likely


Table 1. B. subtilis SpoIIGA–GFP localization patterns.a,b

Strain/spoIIE allele No. of cells % class Ac % class B % class C % class D

PMF 9 wild type 207 31 22 29 18PMF 10 spoIIE64 217 27 18 39 16PMF 14 spoIIE::Tn917VHU7 168 21 13 42 12

a. Cells were harvested 2.5 h after the cessation of logarithmic growth.b. All scoring was performed using cells from several independent experiments using a 60 × objective in conjunction with a 3.33 × side tubemagnifier.c. Class A refers to cells with a disporic signal; class B refers to cells with a monosporic signal; class C refers to cells with unlocalized signal or nodetectable signal; class D refers to cells with aberrant signal (see text). As signal visibility is dependent on correct focus, significantly out-of-focuscells were not scored.


to be of biological relevance (Fig. 2C). We found that theB. megaterium homologue encodes a 307 amino acid(aa) open reading frame (versus 309 aa for B. subtilis),corresponding to a predicted 35 kDa peptide with signifi-cant similarities to the B. subtilis protein (49% identity).This compares with a 42% identity between the B. subtilisand B. thuringiensis proteins (calculated over the available162 aa sequence of the B. thuringiensis carboxyl-ter-minus) and 19% identity between the B. subtilis and C.acetobutylicum proteins. The predicted topological fea-tures of the three proteins are similar (Fig. 2A–C) andare consistent with the model proposed by Stragier et al.(1988).

The sequence alignment also revealed the conservationof several critical bases. The B. subtilis spoIIG49 muta-tion, which causes a 104-fold reduction in sporulation effi-ciency, maps to residue 100 of the B. subtilis sequence(Stragier et al., 1988). This residue (indicated by ‘1’ inFig. 2C) is a perfectly conserved glycine in all of the organ-isms for which we have sequence and is predicted to occurwithin a transmembrane domain in all three organisms.Another perfectly conserved residue is the proline (indi-cated by ‘4’ in Fig. 2C) at position 259 of the B. subtilissequence; strains with a Pro-to-Leu change at this positionhave been isolated as suppressors of a processing-nega-tive allele of pro-sE, suggesting that this amino acid contri-butes to the specificity of the processing reaction (Petersand Haldenwang, 1994). We also note the universal con-servation, beginning at residue 183 of the B. subtilis sequ-ence (indicated by ‘3’ in Fig. 2C), of the Asp-Thr(Ser)-Glycore sequence characteristic of aspartic/retroviral pro-teases, as first noted by Stragier (Pearl and Taylor, 1987;Stragier et al., 1988). This motif occurs in the region ofthe alignment with the greatest overall conservation, andthe only variation in this sequence is the permitted Ser-to-Thr substitution in the second position of the C. aceto-butylicum motif. Aspartic proteases also require a glycinepreceded by two hydrophobic residues positioned approxi-mately 100 bases downstream of the Asp-Thr(Ser)-Glymotif (Pearl and Taylor, 1987). Occurring at residue 279of the B. subtilis sequence (indicated by ‘5’ in Fig. 2C),this glycine is conserved, except in C. acetobutylicum,which has a Gly-to-Ala substitution. In each case, the twopreceding residues are appropriately hydrophobic. Alltopological predictions indicate an extracellular location forthe first six residues of the amino-terminus, as well as forthe large loop between the fourth and fifth membrane-spanning domains. These regions have therefore beenconsidered to be possible interaction sites required forthe transduction of the activating signal to the proteolyticdomain. The alignment in Fig. 2C shows that, while thesix exterior-exposed residues of the amino-terminus arehighly conserved (with the exception of position 2), thelarge extracellular loop between the fourth and fifth mem-

brane-spanning domains is not. This is consistent with arecent report indicating that the entire poorly conservedloop (indicated by a ‘2’ in Fig. 2C) is dispensable forSpoIIGA function (Londono-Vallejo, 1997). This reporthas also shown that a Tyr-to-Ala substitution at residuefour or an Val-to-Ala substitution at residue seven leadsto no decrease in sporulation efficiency. However, an Asp-to-Ala substitution at residue six causes a 2–3 order ofmagnitude inhibition of sporulation in B. subtilis. The effectof an alteration to the conserved isoleucine at residuethree or the conserved leucine at residue five has notbeen evaluated (Londono-Vallejo, 1997). The alignmentprovided here should prove useful for identifying con-served residues to target in future directed-mutagenesisexperiments.

Localization of B. megaterium SpoIIGA–GFP bydeconvolution microscopy

The large size of the B. megaterium cell makes this speciesan attractive model system for protein localization studies.In particular, we hoped to exploit the size of this organismto address the issue of whether SpoIIGA is targeted to theentire septum or only to a peripheral annulus. We wereable to approach this problem by using wide-field deconvo-lution microscopy, a technique that requires taking multipleoptical sections through a cell using standard epifluores-cence optics, then using computational methods to removeout-of-focus and artefactual information from each image(Agard and Sedat, 1983; Agard, 1984). Optimized datasets may then be used to create projections of the samplefrom any perspective. A sequence of many such projec-tions is used to generate a movie simulating rotation ofthe sample around an arbitrary axis. With this in mind,we constructed a spoIIGA–GFP translational fusion in B.megaterium analogous to our B. subtilis construction PMF9. This construction used the intensity-enhanced F64L,S65T variant of GFP (Cormack et al., 1996). PMF 50yielded good results in conjunction with standard micro-scopic techniques and showed septal localization ofSpoIIGA–GFP in >85% of cells. However, the 60 or morelong exposures required for collecting a data set suitablefor deconvolution microscopy required an alternative toGFP providing a more favourable signal-to-noise ratioand less photobleaching. It has been determined recentlythat the IFM technique is sensitive enough to localize pro-teins with as few as 100 molecules per cell (Weiss et al.,1997). We therefore used a modification of the IFM techni-que of Pogliano and Harry (Pogliano et al., 1995) thatemployed a primary antibody raised against GFP and asecondary antibody conjugated to the fluorophore OregonGreen 488. When used in conjunction with an antifadereagent, we were able to obtain fluorescent signal of anintensity and stability sufficient for the collection of




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deconvolution data sets. Figure 1(D–F) shows that the B.megaterium homologue of SpoIIGA is also localized to thesporulation septum. However, the level of detail that weobtained with this technique is best appreciated in three-dimensional projections; QuickTime format movies ofthese and other images can accessed on the MolecularMicrobiology Web site (http:/ /www.blackwell-science.com.products/journals/mole.htm). One insight provided bydeconvolution microscopy is that the initially diffuse fluor-escent signal detected at the onset of SpoIIGA–GFPexpression is preferentially membrane associated. It istherefore likely that SpoIIGA targeting involves an initialnon-specific interaction or insertion of the hydrophobicdomain of SpoIIGA into any membrane it contacts. Inthis model, newly synthesized SpoIIGA rapidly becomesassociated with membrane, then diffuses laterally until con-tacting some other septum-specific protein or componentof the septasome. The interaction of SpoIIGA with thisunknown factor would sequester or anchor SpoIIGA intothe nascent septal structure. While passive, this mechan-ism for SpoIIGA targeting could, nevertheless, result in arapid preferential accumulation of SpoIIGA in the septum.This general model is supported by Fig. 1G, which is acomputer-reconstructed projection down the long axis ofa fluorescently stained PMF 50 cell. At the earliest timepoints after localized signal began to develop, such projec-tions revealed that the localization of signal throughout theseptum was often incomplete (60% of cells), with the mostintense signal being observed in an annulus at the cell per-iphery and with the centre of the cell exhibiting very little orno signal above background levels (Table 2, and Fig. 1G).This pattern also occurs in cells that appear to have com-pleted translocation of DNA into the prespores (approxi-mately 40% of the subclass of cells with an annularpattern) and have therefore presumably finished con-structing a polar septum. The annular pattern of localiza-tion is not an artefact of fixation; PMF 50 cells grownuntil T5 have invariably progressed to a disc-like patternof localization throughout the septum (Fig. 1H). Thedisc-like pattern was distinguished by the significant distri-bution of signal throughout the septum, with the region ofhighest intensity staining being located in the centre ofthe septum.

Localization of B. megaterium SpoIIE–GFP

We have also observed an initial annular localization pat-tern in cells of immunofluorescently stained spoIIE–GFPB. megaterium strain PY1272 (data not shown). However,the progression of localization after the annular stage dif-fers from that seen in PMF 50. Unlike PMF 50 cells,PY1272 cells sporulate at normal frequency and conse-quently pass through an engulfment stage. As shown inthe leftmost cell visible in Fig. 1I, SpoIIE localization at

early times appears as a band across the cell at the siteof septum formation (Barak et al., 1996). At later times,both immunofluorescence and GFP micrographs (right-most cell of Fig. 1I and J respectively) show that theengulfment of PY1272 prespores leads to the formationof a cage surrounding the prespore (QuickTime moviesillustrating the three-dimensional nature of the cage areavailable at the Web site). We also observed the formationof cages in a B. subtilis spoIIE–GFP strain constructed inthis laboratory (data not shown) and found that cages inboth of these strains are stable for several hours. It shouldbe noted that this observation of SpoIIE stability contradictsresults from other groups; it has been reported previouslythat SpoIIE is quickly removed from the sporangium, firstfrom the prespore distal division site, followed shortlythereafter by its removal from the prespore pole (Arigoniet al., 1995; K. Pogliano et al., 1997). While it is possiblethat the GFP tag may confer resistance to degradation,the normal sporulation of this strain implies that the disap-pearance of SpoIIE from the forespore is not required forthe establishment of cell-specific gene transcription.

Implications for models of protein targeting

The septal localization of SpoIIGA in B. subtilis spoIIEstrains with ‘straight and thick’ septa argues against anymodel proposing that initial targeting is delayed until thestage of murein removal. It therefore seems unlikely thatthe targeting of proteins to the polar septum dependsupon recognition of the characteristic double-membranetopology of normal sporulation septa. As the GFP fusionconstructs effectively separate spoIIGB from the spoIIGpromoter, we also conclude that SpoIIGA localizationdoes not depend on the prior localization of pro-sE, whichis itself localized at the spore septum until it is processed(Ju et al., 1997). The localization of SpoIIGA in spoIIEand spo0H backgrounds also indicates that localization


Table 2. Initial appearance of septum in projections down the longaxis of PMF 50 cells processed for IMF.a,b

Localization Number Percentage

Annular septaDNA translocating 7 35Done translocating 5 25

Subtotal 12 60

Disc-like septaDNA translocating 0 0Done translocating 8 40

Overall totals 20 100

a. This table reflects the appearance of the septa approximately1.5 h after the cessation of logarithmic growth and indicates whetheror not translocation of DNA into the prespore appeared to be takingplace. By T5, the septa become uniformly disc-like, and translocatingchromosomes are very rare.b. See Experimental procedures for details.


does not depend on any sF- or sE-dependent transcrip-tion. We have not attempted to determine the localizationof SpoIIGA in the context of spo0A mutations, and it willbe interesting to determine if targeting takes place incells that fail to reposition the FtsZ ring. The observationin B. megaterium of an initial annular pattern of SpoIIGAand SpoIIE localization appears to be consistent with theobservation of Levin et al. (1997) that SpoIIE is co-local-ized with FtsZ rings in a B. subtilis divIC strain grown attemperatures non-permissive for septum formation. Thenext obvious goal is to determine whether SpoIIGA recog-nizes some mark at future sites of septation, or if SpoIIGAlocalization depends on some protein (such as FtsZ, FtsAor a possible ZipA homologue) known or suspected to be acomponent of the septasome.

It is interesting to speculate on the reasons why anannular pattern of SpoIIGA localization is sometimes obser-ved in B. megaterium cells in which DNA translocation hasapparently been completed. It is possible that the co-ordina-tion of septum construction and DNA translocation is differ-ent in this species than in B. subtilis, and DNA translocationmay be essentially completed before the closure of thesporulation septum. However, there are other possibilities.Although it seems unlikely, exclusion of SpoIIGA from thecentre of the annulus might be caused by the steric hin-drance of SpoIIGA by another protein complex. SpoIIIE,which acts as a DNA translocating pore in B. subtilis (Wuand Errington, 1994; Wu et al., 1995), is a possible candi-date. It is also possible that the transition from annular todisc-like localization is triggered by the presence of a laterappearing protein. For instance, the transition may requirethe production and membrane translocation of SpoIIR. Wehave also considered that, while localization to the annulusdoes not depend on the removal of murein from the sep-tum, the transition to the disc-like pattern of localizationmay not occur until murein degradation has taken place.All of our SpoIIGA–GFP fusion strains are abortively dis-poric, but we do not believe that this influences localiza-tion. In B. subtilis, septal SpoIIGA–GFP localization isfirst observed at times corresponding to the initiation ofSpoIIGA synthesis, and well before any unusual accumu-lation of SpoIIGA could occur. Although the timing ofspoIIG expression inB. megaterium has not been described,the appearance of localized protein occurs at the sametime as in B. subtilis. Moreover, a significant proportionof septa examined at the earliest times at which localizedsignal can be observed have already transitioned from theannulus to the disc, suggesting that the kinetics of thistransition are rapid relative to any possible abnormalSpoIIGA–GFP accumulation.

It is important to note that our results also suggest thatthere may be more than one mechanism of targeting.When PMF 9 or PMF 50 cells were grown for >5 h afterthe initial development of fluorescent signal, the GFP

signal remained restricted to the septum. This is in markedcontrast to the localization pattern of SpoIIQ, a sF-depen-dent prespore protein (Londono-Vallejo et al., 1997). Initiallytargeted to the polar septum, SpoIIQ spreads progres-sively around the prespore as engulfment occurs. How-ever, this progression of SpoIIQ distribution occurs evenin a spoIIGB mutant blocked at stage IIi of sporulation,suggesting that SpoIIQ (at least after its initial sequestra-tion at the septum) is targeted differently from SpoIIGA(Londono-Vallejo et al., 1997). Determining the nature ofthe interactions required for protein targeting and under-standing how these proteins are integrated into the septa-some remains a long-term goal.

Experimental procedures

Bacterial strains, culture media, genetic techniquesand in vitro manipulation of DNA

B. subtilis and B. megaterium strains used in this work arelisted in Table 3. E. coli strains MM294 (Bachmann, 1987)and DH5a-mcr (Gibco BRL) were used for routine geneticmanipulations, and E. coli strain S17-I (ATCC 47055) wasused as the donor strain for B. megaterium conjugations(Simon et al., 1983). Isolation of genomic, plasmid and phageDNA, methods for the transformation of B. subtilis and E. colistrains, selection of antibiotic resistance markers and otherstandard genetic manipulations were carried out as describedpreviously (Cutting and Youngman, 1994; Provence and Cur-tiss, 1994). Enzymatic reagents for DNA modification, includingrestriction enzymes, DNA ligase and Taq DNA polymerasewere purchased from Boehringer Mannheim or New EnglandBiolabs and were used as specified by the manufacturers oraccording to standard protocols (Ausubel et al., 1994).

DNA sequencing of the B. megaterium SpoIIGAhomologue

The B. megaterium spoIIGA homologue was identified andsequenced using a PCR approach based on homology to con-served regions in previously identified spoIIGA homologuesand the downstream sigma factor encoded by spoIIGB. Ampli-fication from a QM B1551 chromosomal DNA template usingdegenerate primers (mrBmIIGA2R 58-GGNAAYCARYTNTAY-GAYCC-38 and sigE2.2L 58-ACNGCYTTDATNARNCCDAT-38; DNagency) resulted in a single product of 1.4 kb that wassequenced using the fmol thermal cycling sequencing system(Promega) and determined to be a spoIIGA homologue. Aninverse PCR approach was then used to recover the entirespoIIGA gene. Primers (bmIIGA67Ls: 58-CCTTACCATATC-TGGC-38; and bmIIGB33Rs 58-GCTGTTGATTGAGCG-38)internal to the original PCR product were used to amplify anapproximately 3.0 kb product from a QM B1551 chromosomalDNA template that had been digested with StyI (as determinedby a preliminary Southern blot using the original PCR productas probe against QM B1551 DNA digested with variousenzymes) and self-ligated. This PCR product was T/A clonedinto the SmaI site of pUC19. The resulting 5.7 kb plasmid,



pBm33673, was found to contain the complete spoIIGA homo-logue and was sequenced using a set of nine internal primers.

Construction of SpoIIGA–GFP fusions

B. subtilis spoIIGA–GFP fusions. Using PY79 chromosomalDNA as a template, PCR was used to amplify an 834 bpregion corresponding to the 38 end of the spoIIGA gene. Therightward primer (IIGA391R: 58-TTCATCGGATCCAGTATT-GTCC-38) converts a cryptic BamHI site into a unique BamHIsite. The leftward primer (IIGA1225L: 58-GATAAGGTACCG-ACATTTGCGAACATTTTGAAACG-38) alters the spoIIGAstop codon to a lysine and generates a unique Asp718I site.This product was digested with BamHI and Asp718I andligated into the Asp718I/BamHI backbone of plasmid pKSV7(Smith and Youngman, 1992) to create plasmid pIIGA. TheGFP cassette used in this construction was from pMutGFP, avariant of plasmid TU#65 (Chalfie et al., 1994) incorporatingthe intensity-enhancing S65T mutation (Heim et al., 1995). A740 bp Asp-718I/EcoRI GFP-containing fragment was clonedinto pIIGA, resulting in plasmid pIIGA-GFP. In this plasmid,the spoIIGA fragment is joined to the GFP cassette by alinker encoding KCSQMSVPVGK, the initial lysine of whichwas previously the spoIIGA stop codon. This plasmid wastransformed into strains PY79, PY180 and PY507. Chromo-somal integration and clone selection were as describedpreviously (Barak et al., 1996). All integrations were confirmedby PCR (data not shown). H. Peters (personal communication)has determined by complementation that the GFP fusionprotein is processing negative.

spo0H B. subtilis SpoIIGA–GFP fusions. spo0H ::kan strainM0165 was obtained from P. Stragier (the integrative plasmidused to create this strain is described in Guerout-Fleury et al.,1995). Chromosomal DNA of this strain was prepared andtransformed into PY79 with selection for kanamycin resis-tance. The resulting strain was then transformed with plasmidpWIIGA-GFP. This plasmid is identical to pIIGA-GFP, exceptthat it includes a 155 bp region upstream of spoIIGA requiredfor full expression and duplication of the gene. We amplified aproduct from PMF 9 chromosomal DNA using a rightwardprimer (IIGA104R: 58-GCTTTTTCTAGATCCTCTCATTATA-

CTTCC-38), incorporating a unique XbaI site, in conjunctionwith a leftward primer (GFP-cterm: 58-GGCTGCAGGAAT-TCTACGAATGCTATTTGTATAGTTCATCC-38), incorporat-ing an EcoRI site. This PCR product was digested with XbaI/EcoRI and cloned into pKSV7. Initial transformation andchromosomal integration of this plasmid in the spo0H back-ground used 5 mg ml¹1 chloramphenicol, and the resultingstrain was trained on increasing levels of chloramphenicoluntil a concentration of 80 mg ml¹1 was reached (Janniere etal., 1985; Piggot and Curtis, 1987).

B. megaterium spoIIGA–GFP fusion. PCR was used toamplify a 326 bp product corresponding to the 38 end of the B.megaterium spoIIGA homologue, using QM B1551 chromo-somal DNA as a template. The rightward primer (BmGA21R:58-GTTACAGCGGATCCTTTAAAAGAAATATTGC-38) intro-duced a unique BamHI site. The leftward primer (BmGA347L:58-CGCATCGGTACCGGTGTGAACAAATAAAGG-38) con-verted the spoIIGA stop codon to valine and introduced aunique Asp718I site. This product was digested with BamHIand Asp718I and ligated into the BamHI/Asp718I backboneof plasmid pCON1 (Barak et al., 1996) to create plasmidpBmIIGA. The GFP cassette used in this construction wasfrom pMutGFP2, a variant of plasmid TU#65 (Chalfie et al.,1994) incorporating the intensity enhancing F64L, S65Tmutations (Cormack et al., 1996). The Asp718I/EcoRI GFPcassette from pMutGFP2 was then cloned into pBmIIGA,resulting in plasmid pBmIIGA-GFP. In this construct, thespoIIGA gene fragment is connected to the GFP cassette bya linker encoding VPVQK. This plasmid was conjugated intoQM B1551 as described previously, except that strain S17-Iwas used as the E. coli donor, kanamycin was not used, andonly T7 phage was used for donor counterselection (Barakand Youngman, 1996). Chromosomal integration and cloneselection were as described previously and confirmed byPCR (data not shown).

Preparation of samples for fluorescence andimmunofluorescence microscopy

Strains were grown overnight on Difco sporulation medium(DSM) plates containing 5 mg ml¹1 chloramphenicol and


Table 3. Bacillus strains used in this study.Strain Genotype/phenotype Organism Reference/source

PY79 Prototrophic B. subtilis Youngman et al. (1984)PY180 spoIIE ::Tn917VHU7 B. subtilis Sandman et al. (1987)PY507 spoIIE64 trpC2 thr-5 B. subtilis Barak et al. (1996)PMF 9 spoIIGA–GFP B. subtilis This studya

PMF 10 spoIIGA–GFP B. subtilis This studya

spoIIE64 trpC2 thr-5PMF 14 spoIIGA–GFP B. subtilis This study

spoIIE ::Tn917VHU7M01615 spo0H ::kan B. subtilis P. StragierPMF 37 spoIIGA–GFP B. subtilis This studya

spo0H ::kanPY1272 spoIIE–GFP B. megaterium Barak et al. (1996)PMF 50 spoIIGA–GFP B. megaterium This studya

QM B1551 Wild type B. megaterium P. VaryPS1 Wild type B. megaterium P. Setlow

a. Strains with a spoIIGA–GFP fusion are also spoIIGB, as the integrating plasmid disrupts theoperon.


inoculated into 30 ml of fresh liquid DSM with no antibiotic to adensity of approximately 50–70 Klett units (green filter). Sam-ples were periodically withdrawn and incubated at 48C in pre-servation buffer (Barak et al., 1996). These samples wereeither examined directly for GFP fluorescence or processedfor IFM using the method of Pogliano et al. (1995) with slightmodifications. Initial fixation was in DSM containing 2.5% (v/v)formaldehyde and 0.01% (v/v) glutaraldehyde, and 30 mMNaPO4 buffer. Incubation in lysozyme was for 3 min, and noacetone or methanol was used during subsequent fixation.Labelling reactions used a polyclonal rabbit anti-GFP primaryantibody (Clontech) diluted 1:2000 in conjunction with a goatanti-rabbit secondary antibody conjugated to Oregon Green488 (Molecular Probes) diluted 1:200. Nucleoids were coun-terstained with 2 mg ml¹1 4,6-diamidino-2-phenylindole(DAPI; Sigma). Fluorescent signal from Oregon Green 488was stabilized by treating slides using the SlowFade Lite anti-fade reagent (Molecular Probes) according to the manufac-turer’s instructions. Appropriate control strains (QM B1551and PS 1) that do not express GFP were used. For micro-scopy, we used a customized Olympus IMT2 microscopefitted out with a Nanomotion wide-field deconvolution package(Applied Precision) and equipped with fluorescein isothiocya-nate (FITC) and DAPI excitation and emission filters (Chroma).Images were captured using a cooled CCD (Princeton Instru-ments) interfaced to a Silicon Graphics workstation with Delta-Vision software, which was used to correct, deconvolve andscale data sets of B. megaterium images. Images were typi-cally taken with a 30 s FITC channel exposure followed by a10 s DAPI channel exposure through 30–35 iterations of aprogramme altering the focus by 0.1 mm after each pair ofexposures. Optimized images were then used to createthree-dimensional projections and QuickTime format moviesor flat TIFF format files. Fluorescence micrographs were pre-pared for printing using an Apple Macintosh running ADOBE

PHOTOSHOP 3.05 (Adobe Systems) and printed on a TektronixPhaser IISDX dye sublimation printer.

Acknowledgements

We thank Kelly Dawe and Michael Bender for the use of theirmicroscopes, Howard Peters for his evaluation of pro-sE pro-cessing, and Dave Brown for assistance with computers andin preparing figures for this manuscript. We also thank PatVary and Patrick Stragier for supplying strains, Petra Levinfor providing immunofluorescence protocols, and Imro Barakand Antje Hofmeister for commenting on early versions ofthis manuscript. This work was supported by Public HealthServices grant GM35495 from the National Institutes ofHealth.

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The Bacillus SpoIIGA protein is targeted to sites of spore septum formation in a SpoIIE-independent...

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