Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin

Ole A. Økstad1, Myriam Gominet2, Bénédicte Purnelle3, Matthias Rose4, Didier Lereclus2,5 and Anne-Brit Kolstø1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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PlcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PlcR are: plcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putativeS-layer like surface protein and a putative extracellular RNase. By analysing 37·1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.


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Table 1. Genes surrounding the hbl, plcR and plcA loci

 
Keywords: PlcR regulator, HBL enterotoxin, virulence, Bacillus cereus

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacillus cereus is well established as the aetiological agent of two types of food-borne gastroenteritis giving rise to emesis or diarrhoea, and being caused by the synthesis of emetic toxin or enterotoxins, respectively (reviewed by Turnbull, 1981 ; Granum & Lund, 1997 ). In addition, it currently attracts increasing attention as an opportunistic pathogen in both local and systemic infection (reviewed by Drobniewski, 1993 ). The bacterium is capable of synthesizing a range of membrane-active and tissue-degradative enzymes like phospholipases, haemolysins, cereolysine and a putative collagenase (Drobniewski, 1993 ; Økstad et al., 1999 ).

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.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Bacterial strain and growth conditions.
The B. cereus type strain ATCC 14579, obtained from the American Type Culture Collection (Manassas, VA, USA), was grown at 37 °C in LB broth.

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 5–10 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 11172–11192 of the bc301 clone consensus sequence, while the 3' primer (5'-TAGTATGCCTTGCGCAGTTG-3') corresponded to the reverse complement of positions 53–72 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) . Helix–turn–helix 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 ).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the plcR locus and the plcA upstream region
The 11775 bp bc302 clone contained 10 putative ORFs (Fig. 1a), for which putative functions are listed in Table 1. A putative ORF, encoding a polypeptide of 44 aa of unknown function, has previously been identified immediately downstream of the plcR gene in B. thuringiensis 407 (orf2; Lereclus et al., 1996 ). This organization was conserved in B. cereus ATCC 14579 and the two genes (plcR and orf2) were closely related to those found in B. thuringiensis 407. PlcR has previously been shown to regulate its own transcription (Lereclus et al., 1996 ). Analysis of promoter regions from various PlcR-regulated genes reveals a conserved palindromic sequence TATGNAN4TNCATA which is the putative binding site for PlcR activation (Agaisse et al., 1999 ). Such a plcR box is also present upstream of orf2 and is regulated by PlcR (Agaisse et al., 1999 ), indicating that orf2 and plcR may be transcribed separately.



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Fig. 1. (a) Three chromosomal loci from B. cereus carrying genes regulated by the PlcR transcriptional regulator (bold type) and surrounding genes, covering a total of 37·1 kb. Sloping double lines at the fragment ends indicate incomplete ORFs. bcr1 sequence repeats and putative PlcR-binding sequences are marked. Palindrome sequences predicted to form strong or weak transcriptional regulators are indicated by black and white circles, respectively. DR, direct repeat; IR, inverted repeat. (b) Comparison of organization of genes from the three chromosomal loci in B. cereus ATCC 14579 (genome size 4·28 Mb; Carlson et al., 1996 ) to homologues from B. subtilis 168 (genome size 4·21 Mb; Kunst et al., 1997 ). Three genes, in addition to those under putative PlcR regulation, lacked homologues in B. subtilis (proC, orf6 and orf1). Recognition sites for AscI, NotI and SfiI are indicated by squares, diamonds and arrowheads, respectively.

 
Upstream of plcR, a gene, nprB, resembling neutral proteases from a range of bacterial species is transcribed in the reverse orientation (Fig. 1a). The putative PlcR-binding sequence upstream of the plcR ORF could be utilized in transcriptional regulation of both plcR and nprB (Fig. 1a; Fig. 2). Indeed, it was previously shown that a promoter found upstream of plcR was functional in the reverse orientation (PRP7; Agaisse et al., 1999 ) and its activity was dependent on PlcR. The sequence similarity indicated that B. cereus NprB is a zinc metalloprotease of the thermolysin family (peptidase M4) containing the three amino acids involved in Zn2+ cofactor binding (H397, H401 and E425) and the two conserved active site residues (E398 and H488), strongly indicating that the B. cereus protein is indeed a functional extracellular serine protease.



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Fig. 2. Alignment of the promoter regions of PlcR-regulated genes. The conserved nucleotide sequences found upstream of PlcR-regulated genes are in bold type and the complete palindromic sequence is boxed. This defines the PlcR box which is identical to the sequence previously found upstream of the PlcR-regulated genes isolated from B. thuringiensis (Lereclus et al., 1996 ; Agaisse et al., 1999 ). The transcriptional start sites of the following genes were identified previously: plcR and plcA (Lereclus et al., 1996 ); orf2, nprB and hbl (Agaisse et al., 1999 ). The presumed -10 regions of these promoters are underlined and the transcriptional start site is in bold type. Potential -10 regions of the sfp and cwh gene promoters are also underlined although the transcriptional start site of these genes is not determined.

 
In the regions surrounding nprB, plcR and orf2, seven genes apparently unrelated to PlcR function were identified (Fig. 1a), with the yvfRSTU and ywjDE loci displaying a conserved organization in B. subtilis 168 (Fig. 1b). In B. subtilis, however, the yvfRSTU and ywjDE loci are separated by 317 kb, while ykoW is positioned at 120 °, more than 2 Mb away from yvfRSTU and ywjDE [SubtiList Data Release R14.2, 1998 (www server v2.1.3; http://www.pasteur.fr/Bio/SubtiList.html)]. Three possible factor-independent transcriptional terminator structures were identified when analysing bc302 (Fig. 1a). Although consensus PlcR regulatory sequences were identified in the upstream region of both plcR and orf2 (Fig. 1a), a possible terminator structure could only be identified downstream of orf2, suggesting that the two genes may be co-transcribed.

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 148–471) 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 148–471) 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 helix–turn–helix/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 protein–protein interactions (Table 1). The N-terminal part of Shk1 was of a highly hydrophobic character and was predicted to contain six transmembrane {alpha}-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 5003–5071 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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Fig. 3. Multiple sequence alignment of bcr1 sequence repeats identified in the B. cereus/B. thuringiensis genomes. trp1, ywjE and proC (this paper), bctL (Økstad et al., 1997 ) and bct504a (O. A. Økstad & A.-B. Kolstø, unpublished data) are from B. cereus ATCC 14579; fabZ and celC (Økstad et al., 1999 ) are from B. cereus ATCC 10987; Btwuh is from B. thuringiensis subsp. wuhanensis. The alignment shows three regions of very high sequence conservation. Completely conserved residues are indicated by asterisks, while those found in all sequences but one are indicated by the corresponding letter. Palindrome regions are marked by arrows.

 
Secretory signal sequences
Several gene products under PlcR transcriptional control have been proposed to contain secretory signal sequences, by the examination of charge and hydrophobicity in the N-terminal region (Agaisse et al., 1999 ) using the TOPPRED2 program (http://www.biokemi.su.se/~server/toppred2/) (von Heijne, 1992 ). A more sophisticated prediction method based on neural networks and cleavage site matrices (Nielsen et al., 1997 ) was used in this study to investigate two additional PlcR-regulated genes, cwh and sfp. Amino acid sequences of the 70 N-terminal residues of Cwh and Sfp were submitted to the SignalP V1.1 Prediction Server, Center for Biological Sequence Analysis, Department of Biotechnology, Technical University of Denmark (http://www.cbs.dtu.dk/services/SignalP/). Both proteins were predicted to harbour export signals of 28 and 29 aa, respectively (Table 2). Similarly, the previously examined PRP7 (NprB), Orf2 (PRP14), Nhe (PRP5), PRP6, PRP12, PRP13 and PRP14 (Agaisse et al., 1999 ) were all positive, thereby strengthening the hypothesis of PlcR being a pleiotropic regulator of extracellular virulence factors in B. cereus and B. thuringiensis. The PlcR-regulated proteins previously shown to be secreted (HblC, HblD, HblA, PC-PLC and PI-PLC) were included as positive controls in the signal sequence evaluation (Table 2). In addition, the putative HblB protein, if such a protein is indeed synthesized, was also indicated as being exported, even though the score was lower than for HblA. Of the genes preceded by a PlcR-binding sequence, only PlcR itself was predicted as negative for a signal peptide, as expected from its role as a transcriptional regulator. The role of Orf2 still remains elusive, and the estimated signal sequence for this polypeptide was unusually short for Gram-positive bacteria (http://www.cbs.dtu.dk/services/SignalP/sp_lengths.html) and exhibited lower prediction values than the other PlcR-regulated proteins (data not shown). The nature of Orf2 as a secreted protein is therefore uncertain.


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Table 2. Predicted extracellular transport signals in proteins encoded by PlcR-regulated genes

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Three genomic loci covering a total of 37·1 kb from B. cereus ATCC 14579 and containing several putative PlcR-regulated virulence genes have been cloned and sequenced. Previous studies have shown that more than 10 genes from B. thuringiensis 407 are subject to PlcR transcriptional control (Lereclus et al., 1996 ; Agaisse et al., 1999 ). In this work complete DNA sequences are presented for novel genes putatively controlled by PlcR, in addition to several other apparently functionally unrelated genes in the same chromosomal context.

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 21–70% identity (41–82% 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.


   ACKNOWLEDGEMENTS
 
We are grateful to Frank Kunst for his invaluable help in managing the EC contract. Toril Lindbäck is gratefully acknowledged for providing libraries of B. cereus ATCC 14579 genomic DNA. We wish to thank Anne-Lise Rishovd, Ewa Jaroszewicz, Liv Bjørland and Henning A. Johansen for excellent technical assistance. The work was supported by grants from the Norwegian Research Council to A.B.K. and from Institut Pasteur and Institut National de la Recherche Agronomique to D.L. Funds for cloning and sequencing were supplied by the European Community (EC contract: BIO4-CT96-0655). The project was coordinated by D.L.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Agaisse, H., Gominet, M., Økstad, O. A., Kolstø, A.-B. & Lereclus, D. (1999). PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol Microbiol 32, 1043-1053.[Medline]

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Received 14 April 1999; revised 12 July 1999; accepted 28 July 1999.