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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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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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 Qiagens 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)
.
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RESULTS AND DISCUSSION |
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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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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 510% of total cell protein, its synthesis presumably requires an energy input from the bacteria.
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ACKNOWLEDGEMENTS |
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Received 6 October 2000;
revised 5 January 2001;
accepted 5 February 2001.