8/3/2019 Bacillus 1

1/9

Mole cular Microbiology (1997) 23(6), 1147-1 155

Molecular characterization of the Bacillus anthracismain Slayer component: evidence that it is the majorcel -associated antigenStephane Mesnage, Evelyne Tosi-Couture,* MicheleMock, Pierre Gounon2 and Agnes Fouet*Laboratoire de Genetique Moleculaire des Toxines (URA1858, CNRS), and Station Cenfrale de MicroscopieElectronique, lnstitut Pasteur, 28 rue du Dr Roux, 75724,Paris, Cedex 15, France.SummaryBacillus anthracis, the aetiological agent of anthrax,is a Gram-positive spore-forming bacterium. The cellwall of vegetative cells of B. anfhracis is surroundedby an S-layer. An array remained when sap, a genedescribed as encoding an S-layer component, wasdeleted. The remaining Slayer component, termedEA l, is chromosomally encoded. The gene encodingEA1 (eag)was obtained on two overlapping fragmentsin Escherichia coli and shown to be contiguous to thesap gene. The EA1 amino acid sequence, deduced fromthe eag nucleotide sequence, shows classical Slayerprotein features (no cysteine, only 0.1% methionine,10% lysine, and a weakly acidic pl). Similar to Sapand other Gram-positive surface proteins, EA1 hasthree S-layer-homology motifs immediately down-stream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sapwere co-localized at the cell surface of the wild-typebacilli. However, EA1 was more tight ly bound thanSap to the bacteria. Electron microscopy studiesand in vivo experiments with the constructed mutantsshowed that EA1 constitutes the main lattice of the B.anthracis Slayer, and is the major cell-associatedantigen.

IntroductionBacillus anfhracis, a Gram-positive,terium, is the aetiological agent ofinvolving toxaemia and septicaemia.

spore-forming bac-anthrax, a diseaseFully virulent bacilli

produce two toxins (lethal and oedema toxins) and apoly-y-Dglutamic capsule, encoded by two large plasmids,pXOl and pX02, respectively.The cell wall of 5.antbracisReceived 30 July, 1996; revised 5 December, 1996;accepted 3 Janu-ary, 1997. *For correspondence. E-mail afoue t@p asteur.fr; Tel. (1)45 68 86 54; Fax (1 45 68 89 54.0 1997Blackwell Science Ltd

vegetative cells appears layered n the absence of the cap-sule (Gerhardt, 1967), and fragments displaying a highlypatterned ultrastructure can be observed (Holt and Lead-better, 1969). This structure surrounding he bacterial cellwall is now referred to as the S-layer (for Surface layer).Thus, B. anthracis possesses both a capsule and anS-layer; Bacillus ficheniformis is another bacillus whichhas both of these (Troy, 1973; Tang et al., 1989).

S-layers are proteinaceousparacrystalline sheaths pre-sent on the surface of many Archaebacteria and Eubac-teria (for a review, see Sleytr and Messner, 1983). Mostresult from non-covalent, entropy-driven assembly of asingle (g1yco)protein protomer on the bacterial surface.Generally, a single Slayer is present. Many species haveS-layers, suggesting that the S-layer may be importantin the interaction between the cell and its environment(Sleytr ef a/.,1996). Various functions have been proposedfor S-layers, ncludingshape maintenance, molecular siev-ing, or phage fixation. The Slayer may be a virulence fac-tor, protecting pathogenic bacteria against complementkilling, facilitating binding of the bacterium to host mol-ecules or enhancing its ability to associate with macro-phages (for a review, see Sleytr et al., 1993). S-layers arefound on many bacterial pathogens including Bacteroidesspp., Chlamydia spp., Rickettsia spp., Treponema spp.,Campylobacter spp., Aeromonas spp. and Clostridiumspp. (for compilation, see Messner and Sleytr, 1992).Their possible contribution to virulence therefore seemsworthy of investigation.

We initially hypothesized hat the most abundant proteinfound in the culture supernatants of most B. anthracisstrains is an S-layer component. The chromosomal geneencoding this 94 kDa protein, designated Sap (for surface-rray protein), has been cloned and sequenced, and a nullallelemutant was constructed (Etienne-Toumelinet al.,1995). We report that the surface of the mutant remainsstructured. We describe an analysisof an Slayer constitu-ent, and show it to be the main lattice component, and alsoa major cell-associated antigen.ResultsIsolation of the gene encoding the component of B.anthracis S-layer main lattice in Escherichia coliThe wild type and a Asap mutant strain were examined by

8/3/2019 Bacillus 1

2/9

1148 S. Mesnage et al.electron microscopy. Negatively stained envelope frag-ments were investigated,and a pattern was clearly visibleon both strains (Fig. 1A and IB). This result is not in agree-ment with that previously published (Etienne-Toumelin eta/., 1995). The stability of the S-layer was considerablyimproved in the presence of cations such as Mg2+ duringbacterial resuspension. Preliminary optical diffractionanalysis indicated hat both the wild type and Asap mutantenvelope micrographs gave diffraction patterns, and, there-fore that there is an S-layer on both strains (Fig. 1B).Thus,even in the absence of Sap the cells produce an S-layer.However, the diffraction patterns appeared o be different,suggesting that Sap could be, as previously suggested, anS-layer constituent. We tried to identify the second puta-tive S-layer component. The pellet of the Asap mutant,cured of both virulence plasmids, contained an abundant94 kDa protein (Fig. 2, lane 3). Thus, this protein, whichwe will refer to as EAI was chromosomally encoded. Tostudy this protein further we identified its chromosomalgene and designated it eag.The chymotryptic profile of EA I, stripped from a Asapmutant surface (see the Experimental procedures andFig. 2, lane 3), was different from that of Sap (data notshown). The N-terminal amino acid sequence of EAI wasdetermined (AGKSFPXVPA). It differed from the first 10residues of Sap at one position: a threonine was replacedby a serine residue at position four; the seventh residuecould not be identified with certainty. A degenerated oligo-nucleotide probe, SALl (see the Experimentalprocedures),was deduced from the polypeptide sequence, taking accountof the codon usage bias towards A and T nucleotides in B.anthracis. Total chromosomal digests were probed withSALl in Southern blotting experiments (data not shown),and a size-selected Hindl ll library constructed. A plasmid,pSAL10, hybridizing with SALl was isolated from thislibrary. Part of its restriction map and of that of pEA130(Etienne-Toumelin et a/., 1995) were identical (Fig. 3).This suggested that the newly isolated DNA fragment car-ries a portion of the sap gene. Polymerase chain reaction

(PCR) experiments and sequence data confirmed theoverlap between pEA130 and pSAL10. Southern experi-ments with pSAL10 mapped the SALl sequence to a480 bp EcoRV-Ndel central fragment, just to the right ofthe EcoRV site (Fig. 3). The 820 bp Hincll-Hindlll frag-ment of pSALlO was used as a probe to screen a size-selected Hincl l library, and a second overlapping plasmid,pSAL20, containing he 3' extremity of the gene, was thusisolated.

Sequence analysisThe EcoRV (pSAL1O)-HpaII (pSAL20) fragment (Fig. 3)was sequenced (Accession Number X99724), and shownto carry the entire eag gene, a 2586-nucleotide-long openreading frame (ORF) preceded by a Shine-Dalgarnomotif (AAGGAGG; AG = -17.8 kcal), complementary tothe 16 s ribosomal RNA sequence of B. anthracis (Ashet a/., 1991). It is 722bp downstream from, and in thesame orientation as, sap. There is the standard11nucleo-tide distance between the putative ribosome-bindingsite(RBS) and the ATG codon, observed in other Gram-positive bacteria (Vellanoweth and Rabinowitz, 1992). Notypical -10, -35 sequences were detected. Putative rho-independent transcription erminator sequences, consistingof two palindromic structures, were found 19 and55 nucleo-tides downstream from the stop codon, with calculatedAGvalues of -20.3 kcal and -41.4 kcal, respectively.

The amino acid sequence deduced from the ORF corre-sponds to an 862-residue protein, with a calculated mol-ecular mass of 91 306Da. The polypeptide sequencedetermined from the gel-puri fied protein was found in theamino acid sequence. The position of the N-terminalsequence of the mature protein in the deduced sequencesuggested that the 29 N-terminal residues are a signalpeptide. This sequence possesses the classical featuresof Gram-positive signal sequences, as described bySimonen and Palva (1993). The mature form of EAI con-tains no cysteine, only 0.1% methionine, and 10% lysine,

Fig. 1. Negative staining of E.anthracisSla yer fragments. S-layer fragments obtainedfrom disrupted cells were negatively stainedwith neutralized phospho tungstic acid.A. 9131 (wild-type strain).B. RBA91 (Asap)strain.The bar represents 100 nm.

0 1997 Blackwell Science Ltd, MolecularMicrobiology,23, 147-1 155

8/3/2019 Bacillus 1

3/9

The Bacillus anthracis main S-layer component 1149The 200 N-terminal residues of EA1 and Sap are very

similar (66% identity, 74% similarity; Fig. 4A). This is mainlydue to the presence, in both proteins, of three repeats ofapprox.50 amino acids each, contiguous o the signal pep-tide. These motifs are similar to consensus sequencesdefined by Lupas etal. (1994) as SLH (S-layer homology),and found in many surface proteins of Gram-positivebac-teria. The first motif of each protein shares more sequenceidentity with the other first motif than with the second motifof the same protein and this pattern continues for thedownstream motifs (Fig. 4, A and 6). utside this region,EAI and Sap diverge significantly(19% identity and 30%similarity; Fig. 4A).

EAI is very similar to the S-layer protein (OlpA protein)from Bacillus licheniformis strainNM105 Zhu eta/.,1996).The identity and similarity scores in the 200 N-terminalresidues are 93% and 96%, respectively, and for the restof the protein, the scores are 69% and 78%, respectively(Fig. 4A). Three SLH motifs not described by Zhu et a/.(1996) were found (Fig. 46). No other protein in the databanks was very similar, although the SLH-overlappingregion aligned well with corresponding motifs of otherGram-positive bacterial surface proteins.

Fig. 2. Evidence of an abundant cell-associated 94 kDa protein in aAsap strain. SDS-PAGE of pellet and supernatant fractions ofwild-type (9131) and Asap (RBASI) strains. The pellet fractionswere prepared by boiling the bacteria in Laemmli buffer to strip thesurface proteins. Lanes 1 and 3, boiled pellet; lanes 2 and 4,supernatant.

and has a slightly acidic calculatedpl of 5.43, consistentwith the vast majority of S-layer proteins (Messner,1996).Its hydrophobic amino acid content is 24% which is lowfor an S-layer protein (Sleytr eta/.,1993).

1 kpbFig. 3. Restriction map of some of the plasmids used in this work. The vector for pEA130;pEAI20 pSAL10, and pSAL20 is pUC19, whereasthat for pEA1207, pSAL322 and pSAL303 is pATl13. pEA130, pEA120, and pEA1207 were constructed by Etienne-Toumelin etal. (1995);pSAL10, pSAL20, pSAL322, and pSAL303 were constructed in this work. R5, EcoRV; H3, Hindlll; Hh, Hhal; N, del; H2, Hincll; Hp, Hpall;SpcR,spectinomycin-resistancecassette; ORF sap, ORF of the sap gene; OR F eag, OR F of the eag gene. The long arrows indicate thedirection of transcription; the arrowheads represent the oligonucleotides used to amplify a sequence common to pEA130 and pSALl0.0 1997 Blackwell Science Ltd, Molecular Microbiology, 23,1147-1 15 5

8/3/2019 Bacillus 1

4/9

1150 S. Mesnage et al.

Fig. 4. Protein sequence analysis.A. Schematic comparison of three S-layer components: 5. nthracis EAI and Sap, and B. licheniformis OlpA.6. omparison of the three SLH motifs from EAI , Sap, and OlpA. For each group of motifs, identical residues are represented in black boxes,and conservative changes are shaded. The consensus, as described by Lupas et a /. (1994),s indicated below. The numbers correspond tothe positions of the nearest residues of the deduced amino acid sequences.

Construction, morphological and structural analysesof EA 1-, Sap-, and double-mutant strainsThe function of EA1 could be inve stigated by comparingisogenic wild-type and eagdeleted strains. However, asthe presence of Sap could interfere with the results, weconstructed hree 9131 -isogenic mutants deleted or eithereag (SM91), sap (RBA Sl), or bo th genes (SM11). Thesestrains were obtained by allelic exchange with pSAL322,pEA1207, and pSAL303, respectively (cf. F ig. 3, the fxperi-mental procedures,and Etienne-Toumelinet al., 1995).All morphologicalcriteria of SM91 (Aeag)and wild-typecolonies examined were very similar (flocculationand sedi-mentation properties, bacterium and colony size). In con-trast, these characteristics of RBA91 and S M I 1, similar tothe initial RBA2 Asap mutant (Etienne-Toumelin et a/.,1995), differed from those of the w ild type. The gene rationtimes of the different strains were all similar (40mindoubling time in SP Y m edium).

Negatively stained envelope fragments from the four

strains were examined by electron microscopy (Fig. 1, Aand B, and data not shown). We were unable to obtainan array with the Aeag mutant strain, and no diffractionpattern was observed in the double m utant. These results,together with the presence of an S-layer in the Asapmutant strain, suggest that the presence of A1 is neces-sary an d sufficient for S-layer forma tion in B. anthracis.

Synthesis and localization of the S-layer proteinsThe protein content of cultures of the wild-type and mutantstrains, and the localization of E A1 and Sap, were ana-lysed by SDS-PAGE and Western blotting (Fig. 5). Neitherpellet nor supernatant fractions of S M l l cultures con-tained a detecta ble 94 kDa protein (Fig. 5A). This is con-sistent with the c loned gene en coding the 94 kDa proteinobserved in the RBA91 Asap mutant pellet (Fig. 2). Wetested whether EA1 was synthesized in both the wild-type strain and the Asap mutant. Specific anti-EAl and0 1997 Blackwell Science Ltd, Molecular Microbiotog~3, 147-1155

8/3/2019 Bacillus 1

5/9

The Bacillus anthracis main S-layer component 1151strain, EAI and Sap are both cell bound, and Sap isreleased into the medium.

The localization of EAl and Sap and the relationshipbetween hem were investigatedby immuno-electron micro-scopy (Fig. 6 ) .The specificity of the antibodies used, rabbitanti-EAl and mouse anti-Sap antibodies, was establishedby the absence of binding to the corresponding deletedmutants (data not shown). In the single mutants (Fig. 6,A and B) the remaining protein completely covered thecell surface. On the wild-type surface, the two antibodieswere intermingled (Fig. 6C) . This indicates that neitherof the two proteins, when both synthesized, blocks anti-body access to the other and that they are co-localizedat the cell surface. Presumably, therefore, the wild-typeS-layer could be composedof both proteins.

Fig. 5. Synthesis of the 6. anfhracis S-layer proteins.A. SDS-PAGE of pellet and supernatant fractions of wild-type(9131), Asap (RBASl), Aeag (SM91), and Aeag Asap (SM11)strains. Th e pellet fractions were prepared by sonication. Lanes 1,3, 5, 7, ellet; lanes 2, 4, 6, 8, supernatant.B. lmmunoblot of the same fractions, with rabbit polyclonal anti-EAlantibodies. Lanes as in (A).C. lmmunoblot of the same fractions, with rabbit polyclonal anti-Sapantibodies. Lanes as in (A).

anti-Sap antibodies were obtained, as described in theExperimental procedures. The Western blots (Fig. 5, Band C ) suggested that the antibodies were highly specificas they did not cross-react. EA1 was found in the cell-pellet fraction of both the wild-type and the Asap strains byWestern blotting with the anti-EAl antibodies (Fig. 5B).Therefore, EAl was synthesized by the wild-type strainand was not significantly released nto the medium. How-ever, in contrast to EA1, Sap was present in both fractionsof the wild-type and the Aeag strains. Thus, in the wild-type0 1997 Blackwell Science Ltd, Molecular Microbiology,23,1147-1 155

In vivo expression of EA1 an d SapThe in vivo expression of EA1 and Sap was studied byWestern blot analysis, by testing for antibodies in serafrom mice injected with spores of a strain carrying bothgenes (Fig. 7). The rationale of this experiment is thatantibodies are produced only if the antigen is synthesizedin vivo.A strong signal was obtained against a 94 kDa pro-tein in the wild-type andAsap pellet fractions (Fig. 7 , ane1 and 3), whereas a weak band was seen in the super-natant of the wild-type culture (Fig. 7, lane 2 ) . No signalwas obtained with the Aeag fractions (Fig. 7 , ane 5 and6 ) .Thus the faint signal is most probably due to leakageof EA I into the supernatant. The intense signal evidenceda strong immune reaction to EA1. Thus, EAl is producedin vivo, and is a highly antigenic cell-associated protein.Ezzell and Abshire (1988) described a major extractableantigen which they had termed Extractable Antigen 1(EA1). It is probable that the S-layer component wedescribe and EAl reported by Ezzell and Abshire (1988)are the same and this is why we use the same name.

DiscussionThe surface of the Asap strain (Etienne-Toumelinet a/.,1995) is ordered in arrays. The gene, eag, encoding anS-layer component, termed EA1, was obtained by a two-step procedure, and then disrupted. No S-layer was foundon the surface of the double Aeag Asap mutant, indicatingthat the ea g gene is an S-layer constituent. The eag andsap genes are adjacent on the chromosome; they are inthe same orientation,eag downstream from sap. However,the genes are probably not organized n an operon: there is72 2 bp of non-coding DNA between them and, more con-clusively, EAI is found in abundance in strains in whichthe sap gene has been disrupted. As in the regulatoryregionsof sap and other B.anfhracis genes, no promoterconsensus sequences were found (Etienne-Toumelinel

8/3/2019 Bacillus 1

6/9

1152 S. Mesnage et al.

Fig. 6. Localization by immuno-electron microscopy of EA1 and Sap.A. Asap (RBA91) strain incubated in the presence of rabbit polyclonal anti-EAl antibodies, and 15 nm immunogold-conjugated goat anti-rabbitantibodies.B.Aeag (SMSI) strain incubated in the presence of rabbit polyclonal anti-Sap antibodies, and 15 nm immunogold-conjugated goat anti-rabbitantibodies.C. Wild-type (91 31) strain incubated in the presence of rabbit polyclonal anti -EAl antibodies and mouse polyclonal anti-Sap antibodies, with15 nm immunogold-conjugated goat anti-rabbit and 5 nm immunogold-conjugated goat anti-mouse antibodies. The bar represents 500 nm.

a/ . ,1995). This situation has also been described for theconstitutive promoter of the highly expressed Baci//usbrevis S-layer operon (Adachi et a/.,1989). These genescould be positively regulated, and an activator could com-pensate for the presumably poor RNA polymerase recog-nition sequences.

The deducedEA1 peptide sequence contains hree SLHmotifs. They have been suggested to anchor the protein

subunits to the peptidoglycan or to interfere with bindingto the associated polysaccharides (Lemaire et a/. , 1995;Olabarria et a/ . , 1996). Whatever their precise role, theirpresence together with that of signal peptides in EA 1and Sap is consistent with the localizationof these proteinson the cell surface. However, EAI and Sap do not haveexactly the same distribution: EA 1 is mostly cell bound,whereas Sap is found associated with cells and in the super-natant. This explains the results obtained by Farchauseta/. (1995). The N-terminal sequence they determinedfrom the supernatant fraction is that of Sap; that of theSDS-extracted EA1 could not be determined at position4 or downstream from position 10. We show that Sapand E A I have different residues at these positions. Fromthis, we infer that what Farchaus et a/ . (1995) termedreleased E A I was Sap, and that the cell-associated E A I contained both A 1 and Sap. This correlates well withour data suggesting that plasmid-free strains in vitro pro-duce EA1 and Sap simultaneously.

Our data strongly suggest that the S-layer is produced nvivo. Sera from animals infected with Sterne derivativestend to only recognize the 94 kDa protein produced bySap is either produced n vivo without being immunogenic,

Fig. 7. In vivo expression of the two 8. nthracis S-layercomponents. An immunoblot of pellet and supernatant fractions, ofwild-type (9131), Asap (RBASI) , Aeag (SM91), and Aeag Asap

the Asap but no t the Aeag strain, i.e. EA1. Unlike EA 1(SMII) trains, using pooled sera from mice infected with aSterne-derivative strain. The pellet fractions were prepared by theboilina method. Lanes 1, 3. 5 , 7, Dellet: lanes 2, 4, 6.8,

or it is absent in vivo.The electron microscopy data for the Asap and the-supernatant double-mutant surfaces demonstrate that EA 1 is an0 997 Blackwell Science Ltd, Molecular M/crob/o/ogx 3,1147-1 155

8/3/2019 Bacillus 1

7/9

The Bacillus anthracis main S-layer component 1 153Oligonucleotide probe and DNA librariesThe degenerated oligonucleotide probe (SA L1) deduced fromthe N-terminal sequence of EA1 was 5-GC(TA)GGIA AATC-(TA)TT(TC)CC( TA)GT( TA)GT( TA)CC( TA)GC-3. To con-struct DNA libraries, genomic DNA from strain 9131 wasdigested to completion with H indl ll and H incll. Fragments ofconvenient size were gel purified using the Gene Clean kit(BiolOl Inc.) and ligated into a compatible site of pUC19.The Hindlll and Hincll libraries contained 1300 and 1600independent clones, respectively.

S-layer component. Th e Asap mutant and the wild-typestrain occasionally exhibit different diffraction patternssuggesting that Sap could also contribute to the S-layerarray. We tentatively suggest that EA1 constitutes themain lattice. As no array was observed o n the surface ofth e Aeag strain, it is not clear whether in the presence ofboth proteins there are one or two S-layers. There couldbe a single layer, presum ably with a p attern that is differ-ent from that made by E A I alone. Alternatively Sap m ayform its own, more fragile, structure which needs theE A l array for stabili ty. There are cases where tw o abun-dant surface proteins are synthesized simultaneously. Insome, e.g. Clostridium perfringens (Takumi et a/., 1991),the S-layer is composed of more than one protein. Inother organisms, such as Aquaspirillum serpens (Kistand Murray, 1984), Clostridium difficile (Kawata et a/.,1984) and B. brevis (Tsuboi et a/., 1986), there appear tobe two superimposed S-layers. In other ba cteria, includingBacillus stearothermophilus (Sara et al., 1996) and Cam-pylobacter fetus (Tumm uru and Blaser, 1993), more thanone S-layer can appear sequentially. Image analysis willbe carried out to determine precisely the lattice para-meters of the 9131 and RBA91 arrays.

Our results indicate that EA1 and Sap are expresseddifferently in vivo. The respective contribution of EA1 ,and/or Sap to virulence is under investigation.

Experimental proceduresBacterial strains, vectors and mediaE. coli TG1 (Maniatis et a/., 1982) was used as a host forpUC l9, M13mp18, and M13m p19 derivatives (Yanisch-Perroneta/.,1985). E. coli strain JM83 harbouring pRK24 (Trieu-Cuoteta/.,1991) was used for mating experiments. All B. anthracismutant strains constructed in this study were 9131 derivatives(Etienne-Toumelin et a/., 1995). E. coli cells were grown inLuria (L) broth or on L-agar plates (Miller, 1972). B. anthraciscells were grown in brain-heart infusion medium (Difco labo-ratories) or in SPY medium (Etienne-Toumelin et a/., 1995).Antibiotics were used at the following concentrations: ampicil-lin, 100 pg ml-, and kanamycin, 40 p g ml- for E. coli; andspectinomycin, 60 pg m l , or both E. coli and B. anthracis.DNA manipulations and sequencingMethods for plasmid extraction, endonuclease digestion,Klenow treatment, ligation and agarose gel electrophoresiswere as described by Maniatis et a/. (1982). ChromosomalDNA was extracted as described by Fouet and Sonenshein(1 990). Sequences were determined either from single-stranded DNA (recombinant M13mpl8 or M13mpl9) usingthe dideoxy chain-termination procedure (Sanger et a/.,1977) or from double-stranded DNA (recombinant pUC19)using the PRISM AmpliTaq Dye Primer sequencing kit(Applied Biosystems) with an ABI PRISM 373A sequencer.Each strand was sequenced at least once.0 1997 Blackwell Science Ltd, Molecular Microbio/ogy,23, 147-1 155

Hybridization techniquesColony hybridization and Southern blotting were conductedas described by Maniatis eta/. (1982). DNA fragments andoligonucleotides were radiolabelled by nick translation andterminal deoxynucleotidyl transferase, respectively (Maniatiseta/., 1982).

Disruption of eag and sa p genesRecombinant suicide plasmids were transferred from E. colito B. anthracis by a heterogramic conjugation procedure(Trieu-Cuot et a/., 1987). Allelic exchange was carried outas described previously (Pezard etab, 1991), using the spec-tinomycin-resistance cassette (Fig. 3).To construct a sap-inactivated strain isogenic to 9131,pEA1207 was used (Etienne-Toum elin eta/,, 1995). The eaggene was disrupted with pSAL322 which was constructedas follows. The 1.41 kbp Hindlll-EcoRV fragme nt of pEA130(Etienne-Toumelin et a/., 1995) was ligated into PAT1 13(Trieu-Cuot etal., 1991) digested with H indlll and Smal, giv-ing pSAL320. pSAL201 was obtained by inserting into theSrnal site of pUC19 the blunt-ended 1.14 kbp EcoRV-Ndelfragment of pSAL20 (see the Results). The Hincll-EcoRIfragment of pSAL201, overlapping the 1.14 kbp EcoRV-Ndelfragment of pSAL20, was then transferred into pSAL320 pre-viously cut with Sacl, treated with K lenow and digested withEcoRI, thus creating pSAL321, which harbours the Hindlll-EcoRV fragment of pEA130 and the EcoRV-Ndel fragmentof pSAL20. The Hincll-Sm al fragment from pUC1318 Spc(Murphy, 1985; P. Trieu-Cuot, personal communication) con-taining the spectinomycin-resistance cassette was insertedinto the blunt-ended BarnHl site of pSAL321 to givepSAL322, carrying the deleted eag gene. The plasmid con-taining the double inactivation of eag and sap, pSAL303,was constructed as follows. The 0.75 kbp Hindlll-Hhal frag-ment of pEA120 (Etienne-Toumelin e tab, 1995) was insertedinto pUC19 digested with H indl ll and Srnal,giving pEA1225.The Hindlll-EcoRI fragment of pEA1225 was inserted betweenthe Hindllland EcoRl sites of PAT1 13, giving rise to pEA1226.pSAL211 was obtained by ligating the Xbal-BarnHI fragmentfrom pU Cl31 8 Spc containing the spectinomycin-resistancecassette between the Xbal and BarnHl sites of pSAL201,which harbours the EcoRV-Ndel fragment of pSAL20. Afterdigestion of pSAL211 with Hincll and EcoRI, the 2.45kbpfragment containing the spectinomycin-resistance cassetteand the 3 region of eag was ligated into pEA1226 cut withAcc651, treated with Klenow and digested with EcoRI. The

8/3/2019 Bacillus 1

8/9

1154 S. Mesnage et al.corresponding plasmid, carrying disrupted eag and sapgenes, was designated pSAL303.Protein analysisB. anfhraciscells were grown in SPY medium and harvestedat an ODso0 f approx. 2. Pellets corresponding to 1ml of cul-ture were washed in 50mM Tris-HCI, pH8.0, resuspended inLaemmli buffer (Laemmli, 1970), and boiled for 45 s or soni-cated until complete clarification was achieved. Supernatantswere precipitated with TCA (10% final concentration) andtaken up in 1/10 of their initial volume in Laemmli buffer.Samples (an equivalent of 30pI of pellet and 60 kl of super-natant) were loaded on 10% SDS-PAGE gels. Separatedproteins were transferred onto nitrocellulose sheets usingthe Bio-Rad Trans-Blot system. These blots were probedeither with specific anti-EAl and anti-Sap antibodies, usedat a 1/100000 dilution, or with sera raised against a SterneAlef derivative used at a 1/1500 dilution. The Western blotswere developed using the ECL Western blotting analysis sys-tem (Amersham), diluting the second antibody 1 20000.To digest EA1 by chymotrypsin, the pellet from an RBA2culture (Etienne-Toumelineta/.,1995) was boiled in Laemmlibuffer (Laemmli, 1970) lacking bromophenol blue, and dia-lysed against 20mM Tris-HCI, pH 8.0. The purity of the pro-tein (see, for example, Fig. 2, lane 3) was thus sufficient toestablish a chymotryptic profile. To establish the N-terminalsequence of EAl, the boiled pellet of the RBA2 strain (Eti-enne-Tournelin et a/., 1995) was subjected to SDS-PAGE.The gel was transferred onto diethyl pyrocarbonate (DEPC)and the 94 kDa protein sequenced with an Applied Biosys-tems 470A sequencer equipped with a model 120 phenylthio-hydantoin analyser.Preparation of anti-EA1 and anti-Sap specificantibodies, and of anti-in vivo expressed antigen seraTo obtain rabbit polyclonal antiserum to Sap, a culture super-natant of Aeag mutant was precipitated with 70% ammoniumpersulphate, and subjected to SDS-PAGE. The acrylamideband containing Sap was used to immunize rabbits. Thesame strategy was used to obtain antibodies against EA1: abacterial pellet of a Asap mutant was boiled for 15min inLaemmli buffer, then centrifuged for 10 min at 12000x g.The supernatant, enriched with EA1, was recovered, sub-jected to SDS-PAGE and the region of the gel containingEA1 was cut out. Bouscat rabbits (Charles River) wereinjected subcutaneously with 500 pg of protein, and boostedon days 21 and 36. Rabbits were bled on day 44 and serawere recovered. Mouse polyclonal antiserum to Sap wasobtained by injecting 10 mice with 1pg of the Aeag mutant-culture supernatant used for the rabbits. The mice wereboosted on days 21 and 35 and bled on day 50.To test the in vivoexpression of proteins by the synthesis ofantibodies, six Swiss mice were injected with 10' spores of aSterne Alef derivative. They were sacrificed after 31 d, andtheir sera were pooled.Electron microscopyCells grown to an ODsooof approx. 2.0 were harvested by

centrifugation at 8000 r.p.m., washed in 25 mM Tris-HCI,pH8.0, resuspended in 0.2ml of 20mM Tris-HCI, pH8.0,10 mM MgCI2, n the presence of 30 pl of 425-600 km glassbeads, and disrupted by vortexing for 30s, or by sonicatingfor 10s. They were then fixed with 0.25% glutaraldehyde.Negative staining was performed on 400 copper-mesh gridswith glow-discharged parladion carbon-support film. Gridswere floated on a droplet of the cell-envelope suspensionand then on another droplet of 1% neutralized phospho-tungstic acid. Excess fluid was removed with filter paper.Micrographs were recorded with a Philips CM12, under trans-mission electron microscopy low-dose conditions.Whole mount wild-type, Aeag, Asap, and double-deletedmutant bacteria were analysed by immuno-electron micro-scopy. The cells were fixed in 2% paraformaldehyde, 14mMphosphate buffer, pH7.2. They were then adsorbed onto300 nickel-mesh glow-discharged parladion carbon-coatedgrids. The grids were washed in 14mM phosphate-bufferedsaline, 50mM NH4CI,and incubated on a drop of 14 mM phos-phate-buffered saline, 5% bovine serum albumin, 5% normalgoat serum for 5 min and then in 14 mM phosphate-bufferedsaline, 1% bovine serum albumin, 1% normal goat serum.The antibodies were diluted in the latter solution. Grids wereincubated for 1 h at 25C in a humid chamber, on rabbitanti-EAl antibodies, or on rabbit anti-Sap antibodies, or ona mixture containing rabbit anti-EAl antibodies and mouseanti-Sap antibodies. After four washings on 14 mM phos-phate-buffered saline, the grids were incubated on colloidalgold anti-rabbit or anti-mouse coupled antibodies (British Bio-cell International).

AcknowledgementsWe would like to thank Dr Gervaise Mosser (Institut Curie,Paris) for scientific advice. Edith Duflot, Michel Haustant,and Claude Rolin are acknowledged for protein preparation,technical assistance, and expert help with the photographs,respectively.S.M. was funded by the Ministere de I'Enseigne-ment Superieur et de la Recherche.

ReferencesAdachi, T., Yamagata, H., Tsukagoshi, N., and Udaka, S.(1989) Multiple and tandemly arranged promoters of thecell wall protein gene operon in Bacillus brevis 4. Bac-ferioll71: 1010-1 016.Ash, C., Farrow, J.A.E., Dorsch, M., Stackebrandt, E., andCollins, M.D. (1991) Comparative analysis of Bacillusanthracis, Bacillus cereus, and related species on the

basis of reverse transcriptase sequencing of 16s RNA.ln f J Sys Bacteriol 41:343-346.Etienne-Toumelin, I., Sirard, J.-C., Duflot, E., Mock, M., andFouet, A. (1 995) Characterization of the Bacillus anfhracisSlayer: cloning and sequencing of the structural gene. JBacterioll77: 614-620.Ezzell, J.W., and Abshire, T.G. (1988) Immunological analy-sis of cell-associated antigens of Bacillus anthracis. lnfectlmmun 56: 349-356.Farchaus, J.W., Ribot, W.J., Downs, M.B., and Ezzell, J.W.(1995) Purification and characterization of the major0 1997 Blackwell Science Ltd, Molecular Microbiology, 23, 147-1 155

8/3/2019 Bacillus 1

9/9

The Bacifius anthracis main Sl ay er component 1155changes in S-layer protein synthesis from Bacillus sfearo-fhermophilus PV72 and the S-layer-deficient variant T5in continuous culture and studies of the cell wall composi-tion. J Bacteriol 178: 2108-21 17.Simonen, M., and Palva, I. (1993) Protein secretion inBacillus species. Microbiol Rev 57: 109-1 37.Sleytr, U.B., and Messner, P. (1983) Crystalline surfacelayers on bacteria. Annu Rev Microbiol37: 31 1-339.

Sleytr, U.B., Messner, P., Pum, D., and Sara, M. (1993)Crystalline bacterial cell surface layers. Mol Microbiol 10:Sleytr, U.B., Messner, P., Pum, D., and Sara, M. (1996)Occurence, location, ultrastructure and morphogenesis ofS-layers. In Crystalline Bacterial Cell Surface Protein.Sleytr, U.B., Messner, P., Pum, D., and Sara, M. (eds).New York: Biotechnology Intelligence Unit, AcademicPress, pp. 5-33.Takumi, K., Koga, T., Oka, T., and Endo, Y. (1991) Self-assembly, adhesion, and chemical properties of tetra-gonarly arrayed S-layer proteins of Closfridiurn. J GenAppl Microbiol37: 455-465.Tang, M., Owens, K., Pietri, R., Zhu, X . , McVeigh, R., and

Ghosh, B.K. (1989) Cloning of the crystalline cell wall pro-tein gene of Bacillus licheniformis NM 10. Bacferiol 171:Trieu-Cuot, P., Carlier, C., Martin, P., and Courvalin, P.(1987) Plasmid transfer by conjugation from Escherichia

coli to Gram-positive bacteria. FEMS Microbiol Leff 48:Trieu-Cuot, P., Carlier, C., Poyart-Salmeron, C., and Cour-valin, P. (1991) An integrative vector exploiting the trans-position properties of Tn 7545 for insertional mutagenesisand cloning of genes from Gram-positive bacteria. GeneTroy, F. (1973) Chemistry and biosynthesis of the poly (y-O-glutamyl) capsule in Bacillus licheniformis. II.Characteriza-

tion and structural properties of the enzymatically synthe-sized polymer. J Biol Chem 248: 316-324.Tsuboi, A., Uchihi, R., Tabata, R., Takahashi, Y., Hashiba,H., Sasaki, T., Yamagata, H., Tsukagoshi, N., and Udaka,S. (1986) Characterization of the genes coding for twomajor cell wall proteins from protein-producing Bacillusbrevis 47: complete nucleotide sequence of the outerwall protein gene. J Bacteriol 168: 365-373,Tummuru, M.K.R., and Blaser, M.J. (1993) RearrangementofsapA homologs with conserved and variable regions inCampylobacter fetus. Proc Nafl Acad Sci USA 90: 7265-7269.Vellanoweth, R.L., and Rabinowitz, J.C. (1 992) The influenceof ribosome-binding-siteelements on translational efficiencyin Bacillus subfilis and Escherichia coli in vivo. Mol Micro-

Yanisch-Perron, C., Vieira, J.,and Messing,J. (1985) ImprovedM13 phage cloning vectors and host strains: nucleotidesequences of the M13mp18 and pUC19 vectors. GeneZhu, X. , McVeigh, R.R., Malathi, P., and Ghosh, B.K. (1996)The complete nucleotide sequence of the Bacillus licheni-formis NM105 S-layer-encoding gene. Gene 173: 189-194.

91 1 91 6.

6637-6648.

289-294.

106: 21 27.

bi016: 11 5- 1114.

33: 103-119.

surface array protein from the avirulent Bacillus anthracisDelta Sterne-1. Bacferiol 177: 2481-2489.Fouet, A,, and Sonenshein, A.L. (1990) A target for carbonsource-dependent negative regulation of the citB promoterof Bacillus subfilis. J Bacferioll72: 835-844.Gerhardt, P. (1 967) Cytology of Bacillus anfhracis. Fed ProcHolt, S.C., and Leadbetter, E.R. (1969) Comparative ultra-

structure of selected aerobic spore-forming bacteria: afreeze-etching study. Bacferiol Rev 33: 346-378.Kawata, T., Takeoka, A., Takumi, K., and Masuda, K. (1984)Demonstration and preliminary characterization of a regu-lar array in the cell wall of Clostridium difficile. E M S Micro-biol Lett 24: 323-328.Kist, M.L., and Murray, R.G.E. (1984) Components of theregular surface array of Aquaspirillurn serpens MW5 andtheir assembly in vifro. J Bacteriol157: 599-606.Laemmli, U.K. (1970) Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. NatureLemaire, M., Ohayon, H., Gounon, P., Fujino,T., and Beguin,P. (1995) OlpB, a new outer layer protein of Closfridium

thermocellum, and binding of its S-layer-like domains tocomponents of the cell envelope. J Bacteriol 177: 2451-2459.Lupas, A., Engelhardt, H., Peters, J., Santarius, U., Volker,S., and Baumeister, W. (1994) Domain structure of theAcefogenium kivui surface layer revealed by electron crys-tallography and sequence analysis. J Bacferioll76: 1224-1233.Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecu-lar Cloning: A Laboratory Manual. Cold Spring Harbor,New York: Cold Spring Harbor Laboratory Press.Messner, P. (1996) Chemical composition and biosynthesisof S-layers. In Crystalline Bacterial Cell Surface Protein.Sleytr, U.B., Messner, P., Pum, D., and Sara, M. (eds).Austin, Texas: Biotechnology Intelligence Unit, AcademicPress, pp. 35-76.Messner, P., and Sleytr, U.B. (1992) Crystalline bacterialcell-surface layers. Adv Microb Physiol33: 213-275.Miller, J.H. (1972) Experimenfs In Molecular Genetics. ColdSpring Harbor, New York: Cold Spring Harbor LaboratoryPress.Murphy, E. (1985) Nucleotide sequence of a spectinomycinadenyltransferase AAD (9) determinant from Staphylococ-cus aureus and its relationship to AAD (3) (9). Mol GenGenet 200: 33-39.Olabarria, G., Carrascosa, J.L., Depedro, M.A., and Beren-guer, J. (1996) A conserved motif in S-layer proteins isinvolved in peptidoglycan binding in Thermus thermophi-/us. J Bacterioll78: 4765-4772.Pezard, C., Berche, P., and Mock, M. (1991) Contribution ofindividual toxin components to virulence of Bacillus anfhra-cis. lnfect lmrnun 59: 3472-3477.Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNAsequencing with chain terminating inhibitors. Proc Mat1Acad Sci USA 74: 5463-5467.Sara, M., Kuen, B., Mayer, H.F., Mandl, F., Schuster, K.C.,and Sleytr, U.B. (1996) Dynamics in oxygen-induced

26: 1504-1517.

227: 680-685.

0 1997 BlackwellScience Lid, Molecular Microbiology, 23,1147-1 155