Biotechnology Centre of Oslo and School of Pharmacy, University of Oslo, PO Box 1123, N-0349 Oslo, Norway1
Unité de Biochimie Microbienne, Centre National de la Recherche Scientifique URA 1300, Institut Pasteur, Paris, France2
Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium3
Institut für Mikrobiologie, J. W. Göethe Universität, Frankfurt, Germany4
Unité de Lutte Biologique, Institut National de la Recherche Agronomique, France5
Author for correspondence: Anne-Brit Kolstø. Tel: +47 22 95 84 60. Fax: +47 22 69 41 30. e-mail: annebko{at}biotek.uio.no
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ABSTRACT |
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Abbreviations: PC-PLC, phosphatidylcholine-preferring phospholipase C; PI-PLC, phosphatidylinositol-specific phospholipase C
The EMBL accession numbers for the sequences reported in this paper are given in Table 1.
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INTRODUCTION |
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Several genes encoding putative extracellular virulence factors in entomopathogenic Bacillus thuringiensis, including the HBL and NHE enterotoxins, phosphatidylcholine-preferring phospholipase C (PC-PLC; plc) and phosphatidylinositol-specific phospholipase C (PI-PLC; plcA), are under transcriptional control of a pleiotropic regulator PlcR (Lereclus et al., 1996 ; Agaisse et al., 1999
). The PlcR protein has also been shown to regulate its own transcription and may perform its action by binding to a conserved palindromic DNA sequence upstream of the transcription initiation site of the genes subject to its control.
We have previously sequenced 80 kb from the chromosome of B. cereus ATCC 10987 to compare gene organization with Bacillus subtilis 168 and to study characteristic features of the B. cereus genome (Økstad et al., 1999 ). The type strain of B. cereus, ATCC 14579, has a chromosome map almost identical to that of B. thuringiensis subsp. canadensis HD224 (Carlson et al., 1996
) and has been shown by physical mapping to contain the plcR regulator gene and several putative virulence genes regulated by PlcR (Agaisse et al., 1999
). In addition, it was shown that PlcR from B. cereus ATCC 14579 is functional. The plcR gene and the PlcR-regulated genes are spread over approximately one half of the chromosome and do not form a pathogenicity island. While Bacillus anthracis strain 9131 (Etienne-Toumelin et al., 1994
) harbours a mutated and dysfunctional plcR gene (Agaisse et al., 1999
), B. subtilis 168 does not contain gene homologues to plcR, hblCDAB, plc or plcA [Lereclus et al., 1996
; Kunst et al., 1997
; SubtiList Data Release R14.2, 1998 (www server v2.1.3; http://www.pasteur.fr/Bio/SubtiList.html)].
A major part of the hbl haemolytic enterotoxin locus has been sequenced (Heinrichs et al., 1993 ; Ryan et al., 1997
); however, this sequence did not cover extensive upstream or downstream regions, nor a complete hblB gene. The chromosomal context of other PlcR-regulated genes is also largely unknown and is of great interest with regard to possible operon structures, modes of transcriptional regulation and the presence of mobile genetic elements. To further characterize the PlcR-regulated virulence genes and to investigate their surrounding regions in B. cereus ATCC 14579, we have cloned and sequenced 37·1 kb from the plcR, plcA and hbl loci.
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METHODS |
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Preparation of DNA libraries.
The chromosomal DNA library of B. cereus ATCC 14579 used to clone the hbl locus was constructed in plasmid vector pUC19 using BglII for complete digestion (Lindbäck et al., 1999 ). B. cereus DNA was isolated from 200 ml cultures in exponential-growth phase in LB medium at 37 °C, as described by Lindbäck et al. (1999)
. To clone the 3' end of plcR and its downstream region, total chromosomal DNA from B. cereus strain ATCC 14579 in which plcR was disrupted with an aphA3 cassette, conferring resistance to kanamycin (D. Lereclus, unpublished data), was partially digested with Sau3A and DNA fragments ranging from 510 kb were cloned into the BamHI site of a pUC19 vector. Electroporation of recombinant constructs was performed as described by Chuang et al. (1995)
, using a growth temperature of 18 °C, a 3 min heat shock at 37 °C during cell preparation and 7% (w/v) DMSO for cell storage. Escherichia coli XL-1 Blue MRF' (Stratagene) was used as host. The libraries were plated on LB agar containing ampicillin (50 µg ml-1) for selection of transformants.
Cloning and sequencing of the hbl locus.
Transformants from the library of BglII genome fragments from B. cereus ATCC 14579 were replica-plated in duplicate on nylon membranes (Schleicher & Schuell) and screened by hybridization as described by Kolstø et al. (1990) using a radioactively labelled 3·75 kb genome fragment covering the hblC and hblD genes from B. cereus F0837/76 as probe. The fragment was a kind gift from Patricia Ryan (Rutgers University, New Brunswick, NJ 08903-0231, USA). The screening resulted in the identification of two clones hybridizing strongly with the hbl locus probe (Lindbäck et al., 1999
). The isolated clones, bc300 and bc301, each harboured one BglII fragment, of 9·9 and 11·3 kb, respectively, and were sequenced by subclone construction and by primer-walking. Recombinant plasmid DNA was isolated by using the Qiagen Midi Prep Kit and sequenced by the dideoxy chain-termination method (Sanger et al., 1977
) using the ThermoSequenase Kit (Amersham) and fluorescein end-labelled oligonucleotide primers on an ALF automated sequencer (Pharmacia). Oligonucleotides were prepared at the DNA Synthesis Laboratory, Biotechnology Centre of Oslo, Norway.
The junction region of the non-overlapping clones was subsequently amplified by PCR, using oppositely oriented primers positioned within the hblC coding sequence. The 5' primer (5'-ACCCGACATTAAAATCGTTTG-3') corresponded to positions 1117211192 of the bc301 clone consensus sequence, while the 3' primer (5'-TAGTATGCCTTGCGCAGTTG-3') corresponded to the reverse complement of positions 5372 from the bc300 sequence. The PCR product was cloned in pUC19 as described by Marchuk et al. (1990) and sequenced to obtain a contiguous hbl locus.
Cloning and sequencing of the plcR locus and the plcA upstream region.
The 3' end of plcR and its downstream region was cloned as a 5·5 kb Sau3A fragment by screening recombinant clones for resistance to kanamycin (20 µg ml-1) and was sequenced using a primer-walking strategy. The DNA regions upstream of plcR and plcA and the 5' ends of these genes were cloned and sequenced directly from chromosomal DNA by inverse PCR using appropriate primers. Flanking sequences from known DNA fragments covering the 3' ends and downstream regions of plcR (this paper) and the PI-PLC-encoding plcA gene (Henner et al., 1988 ; Kuppe et al., 1989
; Lechner et al., 1989
; Lövgren et al., 1998
) were scanned for suitable restriction cleavage sites, and chromosomal DNA (5 µg) from B. cereus ATCC 14579 was completely digested with the chosen restriction enzyme(s). The resulting DNA fragments were extracted with phenol and religated in a volume of 200 µl by incubation with 1 U T4 DNA ligase (Roche Diagnostics) for 12 h at 14 °C to preferentially obtain circular molecules. The religated DNA fragments were phenol-extracted and resuspended in 50 µl 10 mM Tris/HCl, 1 mM EDTA, pH 8·0. Close to the ends of the fragments to be extended, two divergent primers were designed and used in PCR, employing 1 µl religated chromosomal fragments as DNA template and cycling conditions as recommended by the supplier of the thermostable DNA polymerases. The amplified DNA fragments were purified (Qiaquick PCR Purification Kit; Qiagen) and sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) as recommended by the supplier, but using half the recommended reaction volumes. Samples were analysed on an automated sequencer (model 377XL; Perkin Elmer). Sequence assembly was performed using the Mac Autoassembler Version 1.4 (Perkin Elmer). After sequence determination, new PCR primers were designed to extend into the unknown regions by inverse PCR. Additional primers were designed to amplify genomic DNA fragments which were also sequenced to exclude artefacts caused by inverse PCR.
DNA sequence analysis.
DNA sequences were analysed using GCG and EGCG programs (Devereux et al., 1984 ). Translated ORFs were used for BLAST and gapped BLAST searches (Altschul et al., 1990
, 1997
) of the non-redundant protein database set at NCBI, USA (http://www.ncbi.nlm.nih.gov/), and for PFSCAN searches (Gribskov et al., 1987
; Lüthy et al., 1994
; Thompson et al., 1994
) of the Prosite and PfamA protein profiles and patterns at ISREC, Switzerland (http://www.isrec.isb-sib.ch/software/pfscan_form.html). Multiple sequence alignments were constructed by PILEUP (Needleman & Wunsch, 1970
; Feng & Doolittle, 1987
; Higgins & Sharp, 1989
). Phylogenetic analysis was performed by CLUSTREE using the neighbour-joining method of Saitou & Nei (1987)
. Helixturnhelix motifs were predicted using HELIXTURNHELIX (Dodd & Egan, 1987
, 1990
) and hydrophobicity was analysed by PEPWINDOW (Kyte & Doolittle, 1982
; Bangham, 1988
). Transmembrane regions were predicted using the TOPPRED2 (von Heijne, 1992
) and DAS (Cserzo et al., 1997
) programs at Stockholm University, Sweden (http://www.biokemi.su.se/~server/), and PHDTOPOLOGY at the PredictProtein Server, EMBL, Germany (http://www.embl-heidelberg.de/predictprotein/). Coiled coil regions were analysed using COILS (Lupas et al., 1991
; Lupas, 1996
) at ISREC, Switzerland (http://www.isrec.isb-sib.ch/software/coils_form.html). Repeats in sequences were identified using COMPARE/DOTPLOT (Maizel & Lenk, 1981
), REPEAT, PALINDROME and STEMLOOP, while factor-independent transcription terminator regions were predicted using TERMINATOR (Brendel & Trifonov, 1984
).
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RESULTS |
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Clone bc303 (4138 bp) covered the region upstream of the PI-PLC-encoding plcA gene. The fragment harboured three genes in addition to 34 bp of the 5' part of plcA (Fig. 1a). B. cereus cwh carried an upstream PlcR regulatory sequence (Fig. 2
) and resembled a range of cell-wall hydrolases of different specificity. The closest relatives were germination-specific N-acetylmuramyl-L-alanine amidases (SleB) from bacilli involved in lysis of the spore-cortex during germination (Table 1
). An SleB protein has been characterized previously from B. cereus (Moriyama et al., 1996
), but this protein was clearly more closely related to B. subtilis SleB than to B. cereus Cwh. Cwh could therefore be a novel subtype of cell-wall hydrolase from Bacillus.
Upstream of cwh, and transcribed in the reverse orientation, is a gene named sfp which could be under PlcR control by employing the same regulatory sequence as cwh (Fig. 1; Fig. 2
). A large region of Sfp (aa 148471) is similar to members of the subtilase family (peptidase family S8; Table 1
), an extensive family of serine proteases whose catalytic activity is provided by a charge relay system similar to proteins from the trypsin family and which contain an Asp-Ser-His catalytic triad (Prosite PDOC00125). A multiple sequence alignment of B. cereus Sfp (aa 148471) with other subtilase members suggested that Sfp contained the catalytic site serine and histidine, and that the aspartate had been conservatively substituted by a glutamate residue, possibly capable of performing an identical catalytic function. B. cereus ATCC 14579 contained sfp directly upstream of plcA. When examining plcA homologues in the databases, the sfp-plcA gene order was conserved in all four plcA loci previously cloned from B. cereus and B. thuringiensis, and Sfp translations, although short and incomplete, were highly conserved, exceeding 95% sequence identity. The DNA sequence identity of the complete sfp-plcA fragments exceeded 97% and was not significantly lower in non-coding than in coding regions.
Analysis of the hbl locus
The complete 21233 bp bc300/301 DNA sequence harboured 14 genes (Table 1), all exhibiting the same orientation (Fig. 1a
). In the hbl upstream region three genes (yndDEF), the organization of which is conserved in B. subtilis 168 [SubtiList Data Release R14.2, 1998 (www server v2.1.3; http://www.pasteur.fr/Bio/SubtiList.html)], encode proteins probably participating in spore germination (Table 1
). The yndDEF, yfnA and sspC genes were spread over 1·3 Mb on the B. subtilis 168 chromosome (Fig. 1b
). That the B. subtilis homologues of the genes surrounding hblCDAB and plcR are arranged differently is not unexpected in light of recent results showing that gene organization is not conserved in the B. cereus ATCC 10987 and B. subtilis 168 genomes (Økstad et al., 1999
).
While trp1, located immediately upstream of hblC, was similar to transcriptional regulators of the helixturnhelix/AraC family (Table 1), a putative PAS- (per, arnT, sim; Nambu et al., 1991
; Ponting & Aravind, 1997
) associated histidine kinase was located downstream of the hblCDAB operon (Fig. 1a
). The protein, encoded by shk1, was similar in the C-terminal part to several kinases from different organisms (Table 1
), including B. subtilis sensor kinases KinA, KinB and KinC involved in sporulation (Trach & Hoch, 1993
; Kobayashi et al., 1995
; LeDeaux & Grossman, 1995
). The two PAS domains and one or more PAC (PAS-associated) domains in the central part (Table 1
) are frequently involved as protein regulatory domains in the sensing of input signals (Parkinson, 1993
; Ponting & Aravind, 1997
). A putative coiled-coil leucine zipper domain located in front of the PAS/PAC domains could be involved in proteinprotein interactions (Table 1
). The N-terminal part of Shk1 was of a highly hydrophobic character and was predicted to contain six transmembrane
-helices, indicating that the protein may be located in the cell membrane.
The hbl locus from B. cereus F0837/76 has been sequenced by Macmillan and co-workers, but the sequence did not cover the complete hblB gene (Heinrichs et al., 1993 ; Ryan et al., 1997
). The organization of the hbl genes was completely conserved in B. cereus ATCC 14579 and the DNA sequence was 97% identical between the two strains. A PlcR box (Fig. 2
) is found upstream of hblC in both strains and transcription of this gene has been shown to be regulated by PlcR (Agaisse et al., 1999
).
A repeated element, bcr1, was identified previously in the B. cereus genome by sequence analysis (Økstad et al., 1999 ). Two of the loci presented in this paper contained bcr1 repeats (upstream of the ywjE, proC and trp1 genes) and the repeats were found exclusively in intergenic regions (Fig. 1a
), as in B. cereus ATCC 10987. bcr1 repeats have been shown by hybridization to be present in strains of B. thuringiensis (Økstad et al., 1999
) and in B. anthracis 7700 (O. A. Økstad & A.-B. Kolstø, unpublished data). A partial bcr1 sequence was identified by database searching in B. thuringiensis subsp. wuhanensis (reverse complement of nt 50035071 in GenBank entry BTU70725) in the vicinity of a cry1Gb1 crystal toxin gene, an IS231F-related transposase and a putative autolysin. So far this repeated element appears to be unique to the B. cereus group of bacteria. An alignment of the identified bcr1 sequence repeats is shown in Fig. 3
.
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DISCUSSION |
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PlcR has been shown to function as a pleiotropic regulator of extracellular virulence factors in B. cereus and B. thuringiensis. The identification of three novel genes, nprB, sfp and cwh, suggested to be regulated by PlcR by virtue of the putative transcription regulatory sequence present upstream of their coding sequences, provided further support to such a notion. Cwh and Sfp were predicted by sequence analysis to function as a cell-wall hydrolase (Cwh) and a subtilase family protease (Sfp), proteins of potential membrane-active and tissue-degradative activity, respectively. Furthermore, a complete sequence of nprB, the B. cereus orthologue of B. thuringiensis PRP7 (Agaisse et al., 1999 ), provided support that this gene encodes a novel potentially degradative neutral protease family member. It is interesting to note that the three novel PlcR-regulated enzymes characterized in this study, although showing significant similarity to proteases (NprB and Sfp) and cell-wall/spore-cortex hydrolases (Cwh) from Bacillus spp., constitute outliers in phylogenetic analyses including sequence-related proteins from the databases (data not shown). Although B. cereus Sfp, Cwh and NprB may not be true sequence or functional homologues of their closest relatives, the conservation of essential amino acids and the high sequence similarities in regions surrounding the catalytic residues strongly support their indicated functions.
The haemolysin BL gene locus is shown by sequencing or hybridization to be present in a number of strains of B. cereus and B. thuringiensis. Several B. cereus strains, however, including ATCC 10987, do not contain the hbl genes. Phylogenetic data describing the relationships between B. cereus strains are insufficient, limiting the possibility to predict whether hbl existed in a last common ancestor of the B. cereus group and was subsequently lost from certain strains, or whether B. cereus acquired the hbl genes at a later stage. A 191 bp region covering the inverted repeat downstream of hblB (Fig. 1a) exhibits 78% DNA sequence identity to a transposase-encoding region highly conserved in two B. thuringiensis plasmids (GenBank D88381 and Y09946; Dunn & Ellar, 1997
) and may suggest that the hbl genes in B. cereus ATCC 14579 have been involved in horizontal transfer processes. It remains to be investigated whether the trp1 and shk1 genes surrounding the hbl operon could constitute a two-component system and whether the genes could have been split by a possible integration of hbl.
A multiple sequence alignment of the deduced HblA (binding component B), HblB, HblC (lytic component L2) and HblD (lytic component L1) protein sequences showed a considerable degree of sequence conservation. By pairwise comparison, HblA, HblB, HblC and HblD showed 2170% identity (4182% similarity), strongly indicating that the hbl operon has arisen by gene amplification from one ancestor gene. A phylogenetic tree constructed by the neighbour-joining method indicated that hblA-hblB constituted the most recent duplication (not shown). Palindrome sequences were identified in both upstream and downstream regions of hblB (Fig. 1a) and might have been implicated in the recombination mechanism. The 91 aa C-terminal part of HblB is not similar to HblA and may be a result of a C-terminal fusion with an ORF during the amplification process. This region was 38% identical to the 49 N-terminal aa of the 246 aa lysin protein from bacteriophage sk1 (Chandry et al., 1997
). Whether this similarity is of functional significance is unclear, given that the function of an eventual hblB gene product is unknown (Beecher & Macmillan, 1991
).
Bacillus cereus, like other bacilli, is well known for its ability to secrete large amounts of proteins into the external milieu. All proteins under putative PlcR transcriptional control, disregarding PlcR itself and perhaps Orf2, were predicted to be synthesized carrying an N-terminal leader signal sequence, promoting their transport to the extracellular environment. These proteins may therefore have the potential to contribute to the virulence of the bacterium in a human or animal host environment, in particular in situations where the bacterium must invade new tissue to increase the nutritional potential. PlcR appears to be a key regulator of these processes and it would be of great interest to investigate possible mechanisms underlying regulation of PlcR activity and whether PlcR is the only protein involved in transcriptional control of the genes carrying an upstream PlcR regulatory sequence.
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ACKNOWLEDGEMENTS |
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Received 14 April 1999;
revised 12 July 1999;
accepted 28 July 1999.