A general strategy for identification of S-layer genes in the Bacillus cereus group: molecular characterization of such a gene in Bacillus thuringiensis subsp. galleriae NRRL 4045

Stéphane Mesnagea,1, Michel Haustant1 and Agnès Fouet1

Toxines et Pathogénie Bactériennes (URA 2172, CNRS), Institut Pasteur, 28 rue du Dr Roux, 75724, Paris cédex 15, France1

Author for correspondence: Stéphane Mesnage. Tel: +44 1603 450514. Fax: +44 1603 450025. e-mail: stephane.mesnage{at}bbsrc.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Despite its possible role in virulence, there has been little molecular characterization of members of the S-layer protein family of the Bacillus cereus group. It is hypothesized that the components of the S-layers are likely to display similar anchoring structures in Bacillus thuringiensis and Bacillus anthracis. Based on inferred sequence similarities, a DNA fragment encoding the cell-wall-anchoring domain of an S-layer component of Bac. thuringiensis subsp. galleriae NRRL 4045 was isolated. The complete gene was identified and sequenced. An ORF of 2463 nt was identified, which was predicted to encode a protein of 821 aa, SlpA. The mature SlpA protein (792 residues) carries three S-layer homology (SLH) motifs towards its amino terminus, each about 50 aa long. These motifs were sufficient to bind Bac. thuringiensis and Bac. anthracis cell walls in vitro by interacting with peptidoglycan-associated polymers, confirming a common wall-anchoring mechanism. The slpA null-allele mutant was constructed and shown to possess no other abundant surface protein. It is proposed that the method described in this paper could be applied to the identification and deletion of any Bac. cereus S-layer gene and is of great value for the molecular and functional characterization of these genes.

Keywords: SLH motifs, cell-wall anchoring, surface antigens, Gram-positive

The GenBank accession number for the slpA sequence is AJ249446.

a Present address: John Innes Centre, Department of Cell and Developmental Biology, Colney Lane, Norwich NR4 7UH, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The cell surface of many archaea and eubacteria is covered by a crystalline array known as the surface layer (S-layer). This macromolecular structure results from the non-covalent, entropy-driven self-assembly of proteins (Sára & Sleytr, 2000 ). Most arrays consist of a single protein species, with one well-documented exception, Clostridium difficile, in which the S-layer consists of two different proteins in equimolar proportion (Takeoka et al., 1991 ; Cerquetti et al., 2000 ). In some species such as Bacillus anthracis, Brevibacillus brevis, Aquaspirillum serpens, Bacillus stearothermophilus and Campylobacter fetus, more than one S-layer protein may be present at the surface of the bacterium. The S-layer proteins may be synthesized simultaneously (e.g. Bre. brevis, A. serpens) or sequentially (e.g. C. fetus and Bac. stearothermophilus) (for review, see Sára & Sleytr, 2000 ). These proteins interact with underlying cell-wall polymers via non-covalent bonds (Boot & Pouwels, 1996 ; Ries et al., 1997 ; Mesnage et al., 2000 ).

During the last decade, several S-layer genes have been cloned and sequenced (for review, see Sára & Sleytr, 2000 ). The deduced primary structures of the proteins encoded by these genes share common features (a very low content of sulphur-containing amino acids, large amounts of aspartic and glutamic acid or a high proportion of hydrophobic residues), but no extensive sequence similarity has been found between them, except in domains involved in cell-wall targeting, i.e. SLH domains (Lupas et al., 1994 ). Therefore, no relationship has been identified between sequence and structural organization of the S-layer. Few data are available concerning the function of S-layers. Nevertheless, some studies have shown that the S-layer is involved in cell integrity and shape maintenance in archaea, in which it is often the only cell-envelope component (Mescher & Strominger, 1976 ; Wildhaber & Baumeister, 1987 ). In some pathogenic bacteria, the S-layer is involved in resistance to complement-mediated killing or interaction with host cells and molecules (Pei & Blaser, 1990 ; Kotiranta et al., 1998 ). In addition, S-layers are often the outermost cell-envelope component and are thought to be involved in the regulation of macromolecule exchange with the environment, by acting as a molecular sieve (Sára & Sleytr, 1987 ; Sára et al., 1992 ).

S-layer proteins are well documented in the genus Bacillus, but not so well in the Bacillus cereus group (for review, see Sidhu & Olsen, 1997 ). The Bac. cereus group comprises four species: Bac. cereus, Bacillus thuringiensis, Bac. anthracis and Bacillus mycoides (Priest, 1993 ). Bac. cereus, Bac. thuringiensis and Bac. anthracis have recently been proposed as a single species (Helgason et al., 2000 ). Some members of the Bac. cereus group are pathogens: Bac. thuringiensis is an entomopathogen commonly used in agriculture, Bac. cereus is a soil bacterium and an opportunistic human pathogen and Bac. anthracis is the causative agent of anthrax, a disease of mammals. Previous studies suggest that the Bac. cereus S-layer may promote interactions with human polymorphonuclear leucocytes, as well as interactions with host (Kotiranta et al., 1998 ). Therefore, S-layer proteins may contribute to the pathogenicity of some bacilli. To date, no S-layer mutant has been produced in the Bac. cereus group, except in Bac. anthracis (Etienne-Toumelin et al., 1995 ; Mesnage et al., 1997 ). In that organism, there are two S-layer proteins, Sap and EA1, and their genes were disrupted independently and simultaneously (Etienne-Toumelin et al., 1995 ; Mesnage et al., 1997 ). However, the function of the S-layer as a virulence factor has not been investigated. Indeed, in Bac. anthracis the S-layer is covered by a polypeptide capsule in vivo, rendering the precise definition of its function difficult (Mesnage et al., 1998 ). To investigate the role of the S-layer in bacteria from the Bac. cereus group, other than Bac. anthracis, we devised, and tested on a given example, a strategy that could be of general use for constructing null mutants in the S-layer-encoding genes, the last step being an allelic exchange. When this work was initiated, only one Bac. cereus group S-layer had been thoroughly studied, that of Bac. thuringiensis subsp. galleriae NRRL 4045, for which biochemical characterization had been reported (Luckevich & Beveridge, 1989 ).

In this paper, we report the cloning of an S-layer gene of Bac. thuringiensis subsp. galleriae NRRL 4045 and the construction of a mutant deficient in S-layer protein synthesis as a means of defining a general method for any Bac. cereus strain. We also analysed the cell-wall targeting mechanism of this protein. We found that the SLH domain interacted with peptidoglycan-associated polymers of Bac. thuringiensis and Bac. anthracis. The S-layer gene cloning and disruption method reported here is general and may be applied to many other Bac. cereus genes encoding S-layer proteins.


   METHODS
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INTRODUCTION
METHODS
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Bacterial strains, vectors and culture media.
Escherichia coli TG1 (Sambrook et al., 1989 ) was used as a host for cloning. E. coli HB101 harbouring pRK24 (Trieu-Cuot et al., 1987 ) was used for mating experiments. Bac. thuringiensis subsp. galleriae NRRL 4045 was provided by T. J. Beveridge (University of Guelph, Canada). In Bac. anthracis SM11, both Bac. anthracis S-layer components have been deleted (Mesnage et al., 1997 ).

For cloning experiments, plasmids pUC19, pNEB193 and pQE30 were routinely used (Yanisch-Perron et al., 1985 ; New England Laboratories; Qiagen). For mating experiments, DNA fragments were inserted into pAT113 (Trieu-Cuot et al., 1991 ).

E. coli was cultured in L-broth or on L-agar plates (Miller, 1972 ). Bac. thuringiensis and Bac. anthracis cells were grown in BHI (Difco) broth or on BHI-agar plates. Antibiotics were used at the following concentrations: 100  µg ampicillin ml-1 and 40 µg kanamycin ml-1 for E. coli; 100 µg spectinomycin ml-1 and 300 µg spectinomycin ml-1 for E. coli and Bac. thuringiensis, respectively.

DNA manipulation and sequencing.
Methods for plasmid extraction, endonuclease digestion, phosphorylation with T4 polynucleotide kinase, ligation and agarose electrophoresis were as described by Sambrook et al. (1989) . PCR amplification and the filling-in of the ends of DNA molecules with Vent DNA polymerase were performed as indicated by the manufacturer of the enzyme. If bacterial colonies were used instead of DNA, the polymerase was added after the initial incubation for 5 min at 100 °C. Chromosomal DNA was extracted as described by Delecluse et al. (1991) . Sequences were determined from PCR products or from double-stranded DNA, by the dideoxy chain termination procedure (Sanger et al., 1977 ) using the prism AmpliTaq Dye Primer sequencing kit (Applied Biosystems) with an ABI PRISM 373A sequencer. Nucleotide and deduced amino acid sequences were analysed using the GCG Wisconsin package (version 9.1; Genetics Computer Group) and the CLUSTAL W program (version 1.8).

General methods.
E. coli cells were made competent as described by Chung & Miller (1988) . Recombinant plasmids were transferred from E. coli to Bac. thuringiensis by heterogramic conjugation (Trieu-Cuot et al., 1987 ). Allelic exchange was carried out as described previously (Pezard et al., 1991 ).

Cloning and disruption of the slpA gene.
The initial DNA fragment (about 620 bp) was isolated by PCR with Vent DNA polymerase, using the oligonucleotides Pst-SLH (Mesnage et al., 1999 ) and Nde-SLH (Table 1). The amplified fragment was inserted into pNEB193 digested with SmaI, giving rise to pSLH4045 (Fig. 1). The fragment was sequenced. A 2·67 kb fragment was then amplified by inverse PCR. Chromosomal DNA was digested with ClaI, for which there is one site in the fragment contained in pSLH4045. It was then self-ligated and used as a template for amplification with the divergent primers BT1 and BT2 (Fig. 1; Table 1). The sequence of the PCR product was determined directly. To sequence the 5' end of the gene, chromosomal DNA was digested with HindIII, for which there was a site in the second fragment amplified, 3' to the fragment from pSLH4045, and inverse PCR was carried out using BT15 and BT19 (Fig. 1; Table 1). A 1·2  kb product was obtained and directly sequenced. To check the DNA sequence encompassing all the cloning oligonucleotides, the 1·3 kb region from the MluI site to 270 bp 3' of BT19 was amplified from chromosomal DNA, using the oligonucleotides BT21 and BT22 (Fig. 1; Table 1), and the PCR product directly sequenced.


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Table 1. Oligonucleotide primers used for PCR and sequencing

 


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Fig. 1. Schematic diagram of Bac. thuringiensis 4045 slpA region. (a) The slpA gene is represented by boxes corresponding to the various protein features. An arrow above indicates the direction of transcription of the gene identified from sequence data. The arrowheads at the ends of vertical dashed lines indicate the position and orientation of the oligonucleotides used for sequencing or cloning experiments described in this work. For clarity, they have been aligned to indicate the fragments obtained using the various pairs. The double bar indicates that the 1·2 kb DNA fragment obtained with primers BT15 and BT19 is not completely represented. (b) Schematic representation of the Bac. thuringiensis chromosomal fragments cloned in various vectors during this work. The names of the plasmids are on the left of the representations, those of the vectors used are in parentheses on the right. The only restriction sites indicated are those referred to in the text: C, ClaI; H, HindIII; M, MluI; S, SapI.

 
A {Delta}slpA strain was constructed as follows. A 2·57 kb fragment, starting with the tenth codon of slpA and ending 135 bp after the stop codon, was amplified from chromosomal DNA using BT23 and BT24 as primers (Fig. 1; Table 1). The fragment was inserted into pAT113 digested with SmaI, giving rise to pBT100 (Fig. 1). pBT100 was digested by SapI and a spectinomycin cassette (Murphy, 1985 ), prepared by digesting pUC1318Spc with HincII, was inserted into it, giving rise to pBT101 (Fig. 1). HB101(pRK24) was transformed with pBT101 and the transformant used in mating experiments with Bac. thuringiensis NRRL 4045 to produce, by allelic exchange, SM4045.

Construction of a Bac. thuringiensis SLH-expression plasmid.
The DNA fragment containing the three SLH motifs was amplified using the oligonucleotides SLHup and SLHdown and inserted into pNEB193 digested with SmaI to give pNSLH100 (Fig. 1; Table 1). pQSLH100 was constructed by digesting pNSLH100 with BamHI and HindIII and inserting the resulting fragment into pQE30 digested similarly (Fig. 1). The plasmid was checked by sequencing.

Protein analysis.
Bac. thuringiensis bacteria were grown overnight in BHI and pelleted by centrifugation. Pellets were washed and resuspended in 1/10 the initial volume of 2 M guanidinium hydrochloride (pH 2·5), as described by Luckevich & Beveridge (1989) to extract specifically the cell-surface-anchored proteins. The samples were then dialysed against Tris/HCl (pH 8·0), resuspended in Laemmli buffer (Laemmli, 1970 ), boiled and 20 µl was loaded onto 10% polyacrylamide gel and subjected to electrophoresis. The gels were fixed and stained with Coomassie brilliant blue.

In vitro interaction of the purified SLH domain with Bac. thuringiensis and Bac. anthracis cell walls.
The His-tagged SLH domain of Bac. thuringiensis was purified according to Qiagen’s recommendations, as described previously (Mesnage et al., 1999 ), with elution at an imidazole concentration of 50 mM. The cell walls of both Bac. thuringiensis NRRL 4045 and Bac. anthracis SM11 strains were prepared as described previously (Mesnage et al., 1999 ). The interaction of the Bac. thuringiensis SLH domain with the cell walls was also studied as described previously (Mesnage et al., 1999 ).

Extraction of polysaccharide, carbohydrate assay and determination of monosaccharide composition.
The total polysaccharide fraction was extracted from cell walls as described by Ekwunife et al. (1991) . Monosaccharide composition was determined by GC on polyol acetate, as described by Sawardeker et al. (1967) .


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Isolation and sequence analysis of the gene encoding the component of the Bac. thuringiensis subsp. galleriae NRRL 4045 S-layer
The first seven amino acid residues of Bac. thuringiensis 4045 mature S-layer protein are identical to the corresponding residues of Sap (Etienne-Toumelin et al., 1995 ; Luckevich & Beveridge, 1989 ). These residues are included in the SLH domain and in fact are, except for one conservative change, also identical to those of EA1 (Mesnage et al., 1997 ). SLH motifs share a high level of sequence conservation. These data suggest that the Bac. thuringiensis S-layer protein starts with an SLH domain. To amplify the SLH motifs, we designed two oligonucleotides, Pst-SLH and Nde-SLH (Mesnage et al., 1999 ; Table 1). An initial fragment was thus amplified, cloned (pSLH4045) and sequenced. Like Sap and EA1, and other Gram-positive S-layer proteins, the Bac. thuringiensis protein harbours three SLH motifs (Mesnage et al., 1997 ; and for review, see Engelhardt & Peters, 1998 ). In two steps of inverse PCR, the DNA fragment overlapping the complete ORF was amplified and sequenced. The corresponding gene was named slpA. Sequencing identified an ORF, encoding a protein with a predicted molecular mass of 87·2 kDa. Analysis of the deduced amino acid sequence showed that there was a 29 aa signal peptide followed by three SLH motifs. The mature protein thus starts, as predicted, with AGKTFPDV (Luckevich & Beveridge, 1989 ) and has a predicted molecular mass of 84·4 kDa, which is similar but not identical to the 91·4 kDa published (Luckevich & Beveridge, 1989 ). A difference between the predicted molecular mass and that deduced from electrophoretic migration has already been observed with Sap and EA1 (Etienne-Toumelin et al., 1995 ; Mesnage et al., 1997 ). Furthermore, in Bac. anthracis, MS confirmed the molecular mass deduced from the sequence for both Sap and EA1 proteins (O. Barzû, personal communication). A pI of 5·33 was calculated from the complete sequence and this is close to the value of 5·0 obtained by isoelectric focusing (Luckevich & Beveridge, 1989 ).

A BLASTP (v. 2.0.14) search was carried out with the complete translation product (SlpA) and with each of the putative domains (i.e. the SLH motifs alone and the protein sequence with these motifs deleted) (Altschul et al., 1997 ). If the complete protein was used, Sap and EA1 were the second and fourth most similar proteins, with a Poisson probability or E value of 3x10-74 and 4x10-73, respectively. If the SLH domain was used alone EA1 and Sap had the second and third most similar SLH domains respectively (E values of 5x10-66 and 3x10-63, respectively). These domains were probably responsible for the high scores obtained with the complete proteins. The CLUSTAL comparison between the SLH domains of S-layer proteins from Bacillus species shows that there is a high level of sequence conservation for the anchoring domain (Fig. 2a). Each motif more closely resembles the equivalent motifs of the other proteins than other motifs of the same protein. If only the putative crystallization domains were compared, protein similarity was observed only with Sap, with an E value of 9x10-9. CLUSTAL analysis was then carried out with the putative crystallization domain of known Bac. cereus S-layer proteins and OlpA from Bacillus licheniformis, which closely resembles EA1 (Fig. 2b) (Mesnage et al., 1997 ; Zhu et al., 1996 ). The alignment of these domains suggests that the sequences of these proteins, within the Bac. cereus group, are more conserved than any two randomly chosen S-layer proteins (Fig. 2b and data not shown). If OlpA and the accompanying Bac. anthracis EA1 sequences were omitted from the comparison, the sequences appeared even more conserved (Fig. 2b, upper cluster). This suggests that the other four proteins compared may have had a common ancestor, closer to that of Sap than EA1. The common sequences conserved in Sap, CTC and SsbC from Bac. stearothermophilus, as described by Jarosch et al. (2000) , are not very well conserved in our alignment, except for the charged residues and lysines in particular, which may reflect the high proportion of these residues in S-layer proteins. Thus, as is often discussed, the sequences of S-layer proteins, even from related organisms (e.g. Bacillus) display little or no identity (Sára & Sleytr, 2000 ). To test whether the sequenced gene was that encoding the S-layer protein described by Luckevich & Beveridge (1989) , we inactivated the gene and analysed the proteins present at the surface of the mutant strain.




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Fig. 2. Similarity between the sequences of SlpA from Bac. thuringiensis 4045 and other Bac. cereus S-layer proteins. (a) SLH anchoring motifs of Bacillaceae S-layer proteins: Bac. thuringiensis subsp. galleriae NRRL 4045 (SlpA_Bt_4045; accession no. AJ249446), Bac. thuringiensis subsp. israelensis 4Q2 (Slp_Bt_TDK; CAA44000), Bac. thuringiensis subsp. finitimus CTC (Slp_Bt_CTC; CAA09981), Bac. anthracis 9131 (EA1_Ba_9131; CAA68063), Bac. anthracis 9131 (Sap_Ba_9131; CAA85408), Bac. licheniformis NM105 (OlpA_Bl_NM105; AAC44405), Bacillus firmus OF4 (Slp_Bf_OF4; AF242295), Bac. stearothermophilus PV72 (SbsB_Bst_PV72; CAA66724), Bacillus sphaericus 2362 (Slp_Bs_2362; P38537), Bac. sphaericus CCM2177 (SbpA_Bs_CCM2177; AF211170), Bre. brevis 47 (MWP_Bb_47; A28555), Bre. brevis HDP31 (HWP_Bb_HDP31; A35129) and Clostridium thermocellum NCIB 10682 (SlpA_Ct; AAC33404). A gap has been left between the first six sequences and the others to indicate the lower level of similarity between the sequences of the SLH motifs of the Bac. cereus group, to which Bac. licheniformis is linked, and the others. (b) Putative crystallization domains of Bac. cereus and Bac. licheniformis S-layer proteins. A gap has been left between the first four sequences and those of EA1 and OlpA to indicate the lower level of similarity between the last two sequences and the others. The order is that generated using the CLUSTAL method. Amino acid residues identical or conserved in at least half the sequences are shown on a black or grey background, respectively.

 
Construction and characterization of an slpA-disrupted mutant
The slpA gene was sequenced directly from PCR products and not cloned. To construct a disruption, we had to clone a fragment overlapping the sequence. This was done by PCR amplification and insertion of the corresponding fragment into a conjugative plasmid (see Methods). A disrupted copy was then constructed by inserting a resistance cassette in the middle of the sequence. By mating E. coli harbouring the construct and Bac. thuringiensis 4045, we obtained a recombinant strain, SM4045, which we then studied. The integration of the plasmid copy at the chromosomal locus had, as expected, occurred by a double crossover event, giving rise to an allelic exchange.

Luckevich & Beveridge (1989) described a specific extraction procedure for the S-layer protein of Bac. thuringiensis 4045. This method, which involves resuspending the cells in guanidinium hydrochloride at low pH as a chaotropic agent, was used on the parental strain and the disrupted SM4045 mutant (Fig. 3 The absence of the 91 kDa protein in the cell-wall fraction of SM4045 confirmed the deletion of the structural gene encoding the Bac. thuringiensis 4045 S-layer protein. It also suggested that no other high-molecular-mass protein was abundant on the surface of Bac. thuringiensis 4045. This contrasts with the situation in other bacteria, including various Bacillus species such as Bac. anthracis or Bac. stearothermophilus, which synthesize two S-layer components. In Bac. anthracis, both proteins are found on the cell surface of the parental strain and in Bac. stearothermophilus, one protein is replaced by another if the medium is changed. Similarly, in other bacteria, a second S-layer protein appears if the gene encoding the S-layer of the parental strain is disrupted (Sára & Sleytr, 2000 ). This striking difference between Bac. anthracis and Bac. thuringiensis 4045 suggests that the S-layer may provide a selective advantage in certain environmental conditions. It is therefore possible that differences in the S-layer reflect different ecological niches or different requirements for the presence of this structure. As the S-layer constitutes 5–10% of total cell protein, its synthesis presumably requires an energy input from the bacteria.



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Fig. 3. Analysis of Bac. thuringiensis 4045 and SM4045 surface proteins. SDS-PAGE of cell-wall fractions of parental (lane 1) and {Delta}slpA mutant (lane 2) strains. The positions of molecular mass markers are indicated on the left.

 
Analysis of the SlpA SLH-motifs as an anchoring domain
Sequencing indicated the presence of three SLH motifs extremely similar to those of Bac. anthracis S-layer proteins. We have previously shown that the SLH motifs of Bac. anthracis are able to bind purified Bac. anthracis cell wall unless the wall is treated to deplete it of the associated pyruvate-containing polysaccharide (Mesnage et al., 1999 , 2000 ). Genetic and biochemical experiments also suggested that similar binding should occur for SLH-containing proteins from various bacteria including Bac. thuringiensis 4045 (Mesnage et al., 2000 ). We tested this conclusion directly. The SlpA-SLH domain was overproduced in E. coli and its capacity for binding to a Bac. thuringiensis cell-wall preparation was analysed (Fig. 4). The SLH domain specifically bound the Bac. thuringiensis cell wall unless the associated polysaccharide was removed by hydrofluoric acid treatment (Fig. 4a). There was also a perfect cross-reaction between Bac. thuringiensis and Bac. anthracis SLH domains and cell walls. Indeed, the SlpA-SLH domain also bound specifically to the Bac. anthracis cell wall (Fig. 4b) and the Sap-SLH domain equally bound to Bac. thuringiensis cell wall (Fig. 4c). In Bac. anthracis, the peptidoglycan-associated polysaccharide binding the SLH domain is pyruvylated and contains galactose, N-acetylglucosamine and N-acetylmannosamine in a molar ratio of 10:3:1. We have previously shown that the cell wall of Bac. thuringiensis 4045 also contains pyruvate (Mesnage et al., 2000 ). PCR co-amplification of an slh sequence and the upstream gene, csaB, the product of which is involved in pyruvylation of the peptidoglycan-associated polysaccharide, gave a positive signal with Bac. thuringiensis 4045 chromosomal DNA (Mesnage et al., 2000 ). We determined the composition of the peptidoglycan-associated polysaccharides from Bac. thuringiensis. Like that of Bac. anthracis, the polysaccharides from Bac. thuringiensis contained galactose, N-acetylglucosamine and N-acetylmannosamine, but they also contained glucose and N-acetylgalactosamine in the molar ratio 6:10:3·5:6:5. Further analyses are therefore required to determine which carbohydrate moiety or moieties are pyruvylated in each of these bacteria, and whether the SLH domains bind to the same ligand.



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Fig. 4. In vitro interaction of the SLH domains of SlpA (a and b) and Sap (c) with purified Bac. thuringiensis 4045 (a and c) or Bac. anthracis SM11 (b) cell wall. Each SLH domain was incubated in the absence (1) or presence (2) of purified cell wall. In (3), cell walls were treated with hydrofluoric acid, thus removing the associated polysaccharide, before incubation with the SLH domains. Lanes: S, soluble fraction (unbound proteins); I, insoluble fraction (bound or self-aggregated proteins).

 
Concluding remarks
We devised and successfully tested a simple method suitable for the cloning and disruption of most, if not all, S-layer genes from the Bac. cereus group. Previous analyses showed that S-layer proteins within the Bac. cereus group are likely to harbour SLH domains and that these SLH domains interact non-covalently with pyruvylated wall-associated polysaccharides (Mesnage et al., 2000 ). Based on these observations, we designed oligonucleotides to amplify DNA fragments encoding the SLH domain from S-layer proteins of bacteria belonging to the Bac. cereus group. The high level of conservation of the SLH sequences, highlighted in Fig. 2(a), indeed confirms that SLH domain sequences of S-layer proteins are highly conserved within the Bac. cereus group, allowing cloning of the corresponding DNA fragment by PCR amplification with the described oligonucleotides. A one- to two-step inverse PCR is sufficient to isolate the rest of the S-layer gene sequence (as in Fig. 1, using ClaI and BT1 and BT2, and then HindIII and BT15 and BT19). The gene, or part of it, is then amplified and inserted into a suicide conjugative vector such as pAT113, which has a broad host range (as in Fig. 1, with BT23 and BT24) (Trieu-Cuot et al., 1991 ). It is not advisable to clone the gene with its regulatory region as this could lead to problems and in many cases has proved impossible (Lemaire et al., 1998 and references therein). The gene is then disrupted by insertion of a selectable marker, such as the spectinomycin resistance cassette inserted into the SapI site in this study, and the copy harbouring the resistance cassette is integrated into the Bac. cereus chromosome at the original locus. The disruption resulting from an allelic exchange due to a double crossover event makes reversion impossible, enabling in vivo studies. Such approaches will be of great value for the molecular and functional characterization of Bac. cereus S-layers, facilitating the comparison of genetically engineered S-layer-deficient isogenic strains. Using degenerate oligonucleotides, taking into account codon usage, this technique could also be applied to many other bacterial species in which genetic transfer is possible.


   ACKNOWLEDGEMENTS
 
We would like to thank Michèle Mock in whose laboratory this work was conducted. We would also like to thank Thierry Fontaine for help with carbohydrate analysis and T. J. Beveridge for sending us the Bac. thuringiensis NRRL 4045 strain. S.M. was a recipient of a ‘Bourse de la Fondation Roux’. This work was supported by DGA 99 34 032/DSP/STTC.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 6 October 2000; revised 5 January 2001; accepted 5 February 2001.