Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation

Cosette Grandvaleta,1, Myriam Gominet1 and Didier Lereclus1,2

Unité de Biochimie Microbienne, Centre National de la Recherche Scientifique URA 2172, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Station de Recherche de Lutte Biologique, Institut National de la Recherche Agronomique, La Minière, 78285 Guyancourt, France2

Author for correspondence: Didier Lereclus. Tel: +33 1 45 68 88 13. Fax: +33 1 45 68 89 38. e-mail: lereclus{at}pasteur.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The immune inhibitor A (InhA) metalloprotease from Bacillus thuringiensis specifically cleaves antibacterial proteins produced by the insect host, suggesting that it may contribute to the overall virulence of B. thuringiensis. The transcriptional regulation of the inhA gene in both B. thuringiensis and Bacillus subtilis was investigated. Using a transcriptional inhA'–lacZ fusion, it was shown that inhA expression is activated at the onset of sporulation. However, the transcriptional start site of inhA is similar to {sigma}A-dependent promoters, and deletion of the sporulation-specific sigma factors {sigma}F or {sigma}E had no effect on inhA expression in B. subtilis. The DNA region upstream from inhA contains two genes encoding polypeptides similar to the SinI and SinR regulators of B. subtilis. SinR is a DNA-binding protein regulating gene expression and SinI inhibits SinR activity. Overexpression of the sin genes affects the expression of the inhA'–lacZ transcriptional fusion in B. thuringiensis: early induction of inhA expression was observed when sinI was overexpressed, whereas inhA expression was reduced in a strain overexpressing sinR, suggesting that inhA transcription is repressed, directly or indirectly, by SinR. inhA transcription was greatly reduced in B. thuringiensis and B. subtilis spo0A mutants. Analysis of the inhA'–lacZ expression in abrB and abrB–spo0A mutants of B. subtilis indicates that the Spo0A-dependent regulation of inhA expression depends on AbrB, which is known to regulate expression of transition state and sporulation genes in B. subtilis.

Keywords: transcriptional regulation, protease, AbrB, Spo0A, Sin

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

a Present address: ENSBANA, Département de Microbiologie, 1 Esplanade Erasme, 21000 Dijon, France.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacillus thuringiensis is a spore-forming Gram-positive bacterium, well known for its entomopathogenic properties, and belonging to the Bacillus cereus group (Schnepf et al., 1998 ). The main insecticidal property of B. thuringiensis resides in the proteinaceous crystal consisting of {delta}-endotoxins (Cry toxins) which are produced during the stationary phase. In addition to {delta}-endotoxins, B. thuringiensis produces phospholipases C, proteases, cytotoxins and other components which may contribute to infection of insects (Heimpel, 1967 ; Salamitou et al., 2000 ; Schnepf & Witheley, 1985 ; Zhang et al., 1993 ). B. thuringiensis is highly resistant to the insect immune system due to its production of two factors, inhibitor A (InA) and inhibitor B (InB), which selectively block the humoral defence system developed by insects against Escherichia coli and B. cereus (Edlund et al., 1976 ).

The InhA protein (previously designated InA) is a metalloprotease presenting similarity with the ‘Bacillus thermoproteolyticus thermolysin, the Pseudomonas aeruginosa elastase and the protease E-15 from Serratia (Lövgren et al., 1990 ). It is also very similar to the protease PrtV of Vibrio cholerae (Ogierman et al., 1997 ). Its amino acid sequence contains the zinc-binding and catalytic active site residues common to these various metalloproteases. In vitro, InhA is secreted and selectively hydrolyses cecropins and attacins, two antibacterial proteins found in the haemolymph of Hyalophora cecropia (Dalhammar & Steiner, 1984 ). InhA is toxic to adult Drosophila (Siden et al., 1979 ).

Most InhA synthesis occurs during stationary phase (Dalhammar & Steiner, 1984 ). It is therefore likely that inhA transcription requires sporulation-specific sigma factors or depends on the complex regulatory mechanisms that control late growth development in Bacillus species. The transcription of many genes induced during the stationary phase is regulated by the product of the spo0A gene. The role in the initiation of sporulation of Spo0A, a DNA-binding protein, has been extensively studied in Bacillus subtilis (Hoch, 1993a ). Upon activation by phosphorylation, Spo0A~P becomes a DNA-binding activator for stage II gene transcription (spoIIA, spoIIE and spoIIG) and a repressor for abrB, a regulator of many post-exponential-phase functions (Strauch & Hoch, 1993 ). The AbrB protein acts as repressor during vegetative growth to prevent expression of stationary-phase-specific genes, including aprE, which encodes the major secreted alkaline protease known as subtilisin. However, AbrB can also activate gene transcription, for example of hpr and competence genes (Dubnau, 1991 ; Perego & Hoch, 1988 ). The sinI–sinR operon also contributes to the regulation of late-growth development in B. subtilis (Smith, 1993 ). SinR, a DNA-binding protein, is a pleiotropic regulator exerting both positive and negative effects on gene expression. Overexpression of sinR inhibits the production of extracellular proteases and represses sporulation by preventing expression of spo0A and stage II sporulation genes (spoIIA, spoIIG and spoIIE) (Gaur et al., 1986 ; Mandic-Mulec et al., 1992 , 1995 ). Inactivation of sinR results in a loss of competence and motility, in a deficiency of autolysin production, and in filamentation (Kuroda & Sekiguchi, 1993 ; Olmos et al., 1997 ). The SinI protein regulates SinR activity by protein–protein interaction, preventing SinR from binding to its target DNA sequence (Bai et al., 1993 ). In B. subtilis, the sin operon has three promoters (Gaur et al., 1988 ). Steady-state levels of RNA initiated at these sites show different patterns relative to each other at different stages of the growth cycle. Expression of sinR is constant throughout the growth cycle, whereas sinI expression is under developmental control, increasing at the end of vegetative growth (Gaur et al., 1988 ). Expression of sinI is stimulated by sporulation activators Spo0A~P and {sigma}H and may be inhibited by the sporulation repressors AbrB and Hpr (Smith, 1993 ; Strauch & Hoch, 1993 ).

The sin operon had not previously been identified in B. thuringiensis. Here we describe two open reading frames, upstream from the inhA gene and transcribed in the opposite orientation, which encode polypeptides similar to the SinI and SinR proteins of B. subtilis. The inhA transcription start site was determined and the regulation of inhA expression was studied in various genetic backgrounds in B. thuringiensis and B. subtilis. The results suggest that the major regulator of inhA expression is AbrB, acting as a repressor of inhA transcription.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains and growth conditions.
The acrystalliferous B. thuringiensis strain 407 Cry- (Lereclus et al., 1989 ) and the B. subtilis 168 trpC2 strain (Anagnostopoulos & Spizizen, 1961 ) were used. The B. thuringiensis 407 strain, in which the spo0A gene is disrupted ({Delta}spo0A strain), has been previously described (Lereclus et al., 1995 ). Escherichia coli strain TG1 [{Delta}(lac–proAB) supE thi hsdD5 (F' traD36 proA+ proB+ lacIq lacZ{Delta}M15)] (Gibson, 1984 ) was used as host for construction of plasmids. Plasmid DNA used to electrotransform B. thuringiensis was prepared from E. coli strain SCS110 [rpsL (Strr) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44 {Delta}(lac–proAB) (F' traD36 proAB lacIq Z{Delta}M15)] (Stratagene). B. thuringiensis strain 407 was transformed by electroporation as previously described (Lereclus et al., 1989 ). B. subtilis was transformed by plasmid or chromosomal DNA as previously described (Anagnostopoulos & Spizizen, 1961 ; Kunst et al., 1994 ). The antibiotic concentrations used for bacterial selection were as follows: ampicillin (100 µg ml-1 for E. coli), spectinomycin (100 µg ml-1 for E. coli), erythromycin (10 µg ml-1 for B. thuringiensis and B. subtilis), kanamycin (10 µg ml-1 for B. subtilis, and 300 µg ml-1 for B. thuringiensis) and chloramphenicol (5 µg ml-1 for B. subtilis). E. coli cells were grown at 37 °C in LB medium. B. thuringiensis and B. subtilis cells were grown at 30 °C in LB medium or in sporulation-specific media: HCT medium was used for B. thuringiensis (Lecadet et al., 1980 ), and SP medium was used for B. subtilis (Lereclus et al., 1995 ). Activation of the xylA promoter in B. thuringiensis was induced by adding xylose (20 mM final concentration) in the culture medium. This concentration of xylose confers a maximal expression of the xylA promoter throughout the growth of cells cultured in HCT medium (unpublished data).

DNA manipulation.
Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturers. Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure using Qiagen kits. Total DNA was extracted and purified from B. thuringiensis and B. subtilis as previously described (Msadek et al., 1990 ). Oligonucleotide primers were synthesized by Genset (Paris, France). PCR was performed using a GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer). Nucleotide sequencing by the dideoxy-chain-termination method (Sanger et al., 1977 ) was performed using double-stranded DNA as the template, the T7 Sequencing Kit from Pharmacia P-L Biochemicals and [35S]dATP{alpha}-S (15 TBq; Amersham). Some nucleotide sequences were determined by Genome Express (Grenoble, France).

Cloning and sequencing of the sinI, sinR and inhA genes.
A 2·4 kb DNA fragment containing sinI, sinR and the 5' end of the inhA gene was cloned from a B. thuringiensis 407 mutant strain carrying a mini-Tn10 transposon in the promoter region of sinI (unpublished data). The pUC replicon in the mini-Tn10 element (Steinmetz & Richter, 1994 ) was used to clone the chromosomal DNA sequences flanking the insertion site. Chromosomal DNA from the mutant strain was digested with HindIII (a restriction enzyme for which no site is present in the mini Tn10 element) and treated with T4 DNA ligase. The ligation mixture was used to transform E. coli cells, and transformants were selected for resistance to spectinomycin. SpcR transformants contained a plasmid with a 2·4 kb chromosomal DNA fragment. Its nucleotide sequence was determined.

The complete inhA gene was cloned by PCR amplification from B. thuringiensis 407, using primers Sin14 and Sin15. Sin14 (5'-CCGGAATTCCCTCTATGGAAATTATAAATTG-3') creates an EcoRI site (underlined), and is complementary to sequences upstream from inhA in the 2·4 kb DNA fragment. Sin15 (5'-CGGGATCCCACTATTTTTATCCAGTTC-3') creates a BamHI site (underlined), and is complementary to sequences downstream from inhA, as deduced from the published nucleotide sequence (Lövgren et al., 1990 ). The PCR product (a 2·7 kb DNA fragment) was purified, digested by EcoRI and BamHI, and inserted into the vector pHT304 (Arantes & Lereclus, 1991 ). The 3' end of the inhA gene from B. thuringiensis 407 was sequenced directly from plasmid DNA. Thus, the nucleotide sequence of a contiguous 3·8 kb DNA fragment was determined (GenBank accession number AF287346).

Plasmid constructions.
The DNA fragment containing the inhA promoter region was amplified by PCR using primers GC2 (5'-CCCAAGCTTATGTAATTCCTCCCTAATTAT-3') and GC3 (5'-CCCGGATCCTCTGAAGCCTTCGAATCAATTACC-3'). The amplified 540 bp DNA fragment was digested with BamHI and HindIII (sites underlined) and ligated between the BamHI and HindIII sites of pHT304-18Z (Agaisse & Lereclus, 1994 ) to produce pHT304{Omega}inhA'–lacZ.

The plasmids pHT315pxyl{Omega}sinI and pHT315{Omega}pxylsinR, overexpressing the sinI and sinR genes, respectively, were constructed as follows. The promoters of the xylA gene and the xylR gene, encoding the transcriptional repressor of xylA, were amplified by PCR from chromosomal DNA of the B. subtilis strain 168, using primers Xyl1 (5'-CACATGCATGCCATGTCACTATTGCTTCAG-3') and Xyl2 (5'-GCTTCTAGATTGAGCCATGTGATTTCCCC-3'). The amplified 1·5 kb DNA fragment was ligated between the SphI and XbaI sites of the high-copy-number plasmid pHT315 (Arantes & Lereclus, 1991 ) to give the plasmid pHT315xyl. The B. thuringiensis sinI gene was amplified by PCR with Sin1 (5'-CGGGATCCCATATTTCCTCCCC-3') and Sin2 (5'-CCGGAATTCGGAAGTTCAGTTAATGA-3'), and the sinR gene with Sin4 (5'-CCGGAATTCCAGATCTTTTAGCTAAAGAC-3') and Sin5 (5'-CGGGATCCAACGTATGAAGCCATCCAGTC-3'). Both amplified fragments were digested by EcoRI and BamHI (underlined) and ligated into the expression vector pHT315xyl. The resulting plasmids were named pHT315xyl{Omega}sinI and pHT315xyl{Omega}sinR, respectively.

Construction of the B. thuringiensis 407 [inhA'–lacZ] strain.
The chromosomal B. thuringiensis inhA gene was disrupted by homologous recombination using the thermosensitive plasmid pRN5101 as the intermediate vector (Villafane et al., 1987 ). This plasmid confers resistance to erythromycin in Gram-positive hosts and resistance to ampicillin in E. coli. To construct the chromosomal inhA'–lacZ transcriptional fusion, the 5' and 3' parts of the inhA gene were amplified by PCR using the primer pairs InA1 (5'-ACCCAAGCTTCAGAGAAAGAAACAAAGAAAGC-3') and InA2 (5'-CGGAATTC-ATGCGCGTTTACACCAACTG-3'), and InA3 (5'-CGGAATTCTGTTATGAGTGGCGGCACGTGG-3') and InA4 (5'-CGGGATCCTTCGCTACACCCGCTAAGCC-3'), respectively. The PCR fragments were digested with appropriate restriction enzymes (underlined) and purified as a 0·9 kb HindIII–EcoRI fragment and as a 1 kb EcoRI–BamH1 fragment, respectively. The promoterless lacZ gene was isolated as a 3·2 kb EcoRI DNA fragment from pHT304B'Z (Lereclus et al., 1996 ). The three DNA fragments were ligated together between the HindIII and BamHI sites of pRN5101. The appropriate orientation of the lacZ gene between the 5' and 3' regions of inhA was determined by restriction analysis of the recombinant plasmids isolated from the E. coli transformants. The selected recombinant plasmid was introduced into B. thuringiensis by electroporation and the replacement of the chromosomal copy of inhA by the transcriptional inhA'–lacZ fusion was obtained as previously described (Lereclus et al., 1992 ). The recombinant strain, designated B. thuringiensis 407 [inhA'–lacZ], was Lac+ and sensitive to erythromycin. The construction was verified by PCR, using appropriate oligonucleotide primers.

Construction of B. subtilis mutant strains containing pHT304{Omega}inhA'–lacZ.
B. subtilis 168 containing pHT304{Omega}inhA'–lacZ was transformed with chromosomal DNA purified from {Delta}sigE, {Delta}sigF, {Delta}spo0A, {Delta}abrB and {Delta}spo0A{Delta}abrB B. subtilis mutants (laboratory stock) (Msadek et al., 1998 ). The recombinant strains were selected for erythromycin and kanamycin resistance ({Delta}sigE, {Delta}sigF and {Delta}spo0A mutants containing pHT304{Omega}inhA'–lacZ), erythromycin and chloramphenicol resistance ({Delta}abrB mutant containing pHT304{Omega}inhA'–lacZ), or erythromycin, kanamycin and chloramphenicol resistance ({Delta}spo0A–{Delta}abrB mutant containing pHT304{Omega}inhA'–lacZ).

RNA extraction and primer extension.
Total RNA was extracted from B. thuringiensis strain 407 Cry-, and the inhA transcription start site was determined by primer extension as previously described (Grandvalet et al., 1999 ) using synthetic oligonucleotide GC1: 5'-TCAGCATATGCAGATTGAGTGCCAGCTCC-3'. DNA sequencing was performed by the dideoxy-chain-termination method with double-stranded pHT304{Omega}inhA'–lacZ plasmid as template and the primer GC1.

ß-Galactosidase assay.
B. thuringiensis and B. subtilis cells containing lacZ fusions were grown in LB or SP medium at 30 °C, harvested by centrifugation for 4 min in an Eppendorf microcentrifuge, suspended in Z buffer (Na2HPO4 . 7H2O, 60 mM; NaH2PO4 . H2O, 40 mM; KCl, 10 mM; MgSO4 . 7H2O, 1 mM; dithiothreitol, 1 mM) and were shaken with 0·4 g acid-washed glass beads (212–300 µm diameter) for 30 s, using a FastPrep FP120 Instrument Savant (BIO 101). Cell debris was eliminated by centrifugation and ß-galactosidase assays were performed as previously described (Msadek et al., 1990 ). The specific activities are expressed in units of ß-galactosidase per mg of protein (Miller units). Protein concentrations were determined using the Bio-Rad protein assay.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the inhA, sinI and sinR genes from B. thuringiensis strain 407
We identified a Tn10-insertional mutation affecting expression of plcR (D. Lereclus & M. Gominet, unpublished results). PlcR is a pleiotropic regulator of virulence gene expression (Agaisse et al., 1999 ; Lereclus et al., 1996 ). A 3·8 kb DNA region surrounding the Tn10 insertion site was obtained as two fragments and sequenced, as described in Methods. It contained three open reading frames (ORFs) each with a putative Shine–Dalgarno sequence at an appropriate distance upstream from a potential start codon (Fig. 1a). Two of these ORFs are small, separated by 82 nucleotides and encoding polypeptides of 44 and 107 amino acids. Their deduced products are similar to SinI (34% identity and 57% similarity) and SinR (66% identity and 83% similarity) of B. subtilis, respectively, and were therefore designated sinI and sinR. The third ORF, transcribed in the opposite direction from sinIsinR, encodes a polypeptide of 795 amino acids highly similar (92% identity) to the InhA protease from B. thuringiensis var. alesti (Lövgren et al., 1990 ). However, the first 109 amino acids of the B. thuringiensis 407 polypeptide are missing from the published sequence of the InhA protein. Despite this difference, we assume that this ORF corresponds to the inhA gene from B. thuringiensis strain 407. The putative coding sequence of inhA starts with an ATG codon preceded at an appropriate distance by a potential ribosome-binding site (GGAGG) (Fig. 1a).



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Fig. 1. Analysis of the inhA promoter region. (a) Genetic organization of the sinR, sinI and inhA genes, and DNA sequence of the intergenic region between sinI and inhA genes. Asterisks indicate transcription start sites. The putative -10 and -35 boxes of the inhA promoter are boxed. The potential ribosome-binding site is shown in bold and the start codons of sinI and inhA (TTG and ATG, respectively) are double underlined. The potential AbrB-binding sites (AT–7 bp–AAT) are underlined. (b) Determination of the inhA transcriptional start site by primer extension assay. Total RNA was extracted from B. thuringiensis cells 3 h (t3) and 4 h (t4) after the end of the exponential phase. RNA was subjected to primer extension with the oligonucleotide GC1 and the same oligonucleotide was used to prime dideoxy sequencing reactions (lanes A, C, G, T).

 
InhA is an exported protein, so we looked for a signal peptide cleavage site by the method described by Nielsen et al. (1997) . The amino acid sequence of InhA was submitted to the SignalP V1.1 Prediction Server, Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/). The most likely cleavage site was found between positions 31 and 32 of the protein (between an alanine and a glutamic acid residue: AYA-ET). Moreover, the size we propose for the inhA coding sequence is confirmed by an alignment analysis: the amino acid sequences of InhA and the PrtV protease from V. cholerae match along their entire lengths (38% identity; 54% similarity).

Determination of the transcription start site and analysis of the inhA gene expression in B. thuringiensis
To localize the promoter region of the inhA gene, the transcription start site was determined by primer extension analysis using oligonucleotide GC1 and RNA extracted from B. thuringiensis cells during the stationary phase (Fig. 1b). Two transcriptional start sites were identified at nucleotide positions -37 and -38, with reference to the presumed translational start codon. A -35 (TTGAAA) and a -10 (TAAAAT) hexamer separated by 17 nucleotides, highly similar to the {sigma}A promoter consensus (TTGACA 17–18N TATAAT), were identified at an appropriate distance from the transcriptional start site (Fig. 1a).

The transcriptional activity of the inhA promoter was first assayed by constructing a transcriptional fusion between the inhA promoter region and the lacZ gene in pHT304-18Z (see Methods). The resulting plasmid (pHT304{Omega}inhA'–lacZ) was introduced into B. thuringiensis 407 and the cells were grown at 30 °C in a sporulation-specific medium (HCT). The ß-galactosidase activity of cells was measured at various stages of growth (Fig. 2a). ß-Galactosidase activity in cells harbouring pHT304{Omega}inhA'–lacZ was relatively low during the exponential phase of growth and increased after the onset of the stationary phase. To check that the plasmid copy number [about four copies of pHT304 per chromosome equivalent (Arantes & Lereclus, 1991 )] did not result in a large alteration of the regulation of inhA expression, the transcriptional inhA'–lacZ fusion was integrated at the inhA locus of the B. thuringiensis chromosome (see Methods). Interruption of the inhA gene did not affect the growth rate of B. thuringiensis cells (not shown). ß-Galactosidase activity of the B. thuringiensis strain 407 [inhA'–lacZ] was measured at various stages of growth in HCT medium (Fig. 2b). The level of inhA-directed ß-galactosidase synthesis was very weak during exponential growth and increased at the onset of the stationary phase. Although the activity level was lower for the chromosomal than for the plasmid construction (40 Miller units for the chromosomal fusion at t0 versus 900 Miller units for the pHT304{Omega}inhA'–lacZ plasmid), the time course was similar: in both cases inhA expression was activated during the stationary phase. This validated the use of the plasmid-borne and chromosomal transcriptional fusions to investigate the regulation of inhA expression in various genetic backgrounds.



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Fig. 2. Expression of inhA'–lacZ transcriptional fusions in B. thuringiensis strain 407: ß-galactosidase activity of (a) B. thuringiensis strain 407 carrying the plasmid pHT304{Omega}inhA'–lacZ and (b) B. thuringiensis 407 [inhA'–lacZ]. The cells were cultured at 30 °C in HCT medium. Time zero indicates the onset of the stationary phase (i.e. when the slope of the growth curve starts to decrease).

 
Effect of sinI and sinR overexpression on the inhA regulation
Since the putative inhA promoter resembles a promoter recognized by the E{sigma}A form of RNA polymerase, the induction of inhA expression at the onset of the stationary phase may be due either to the disappearance of a repressor active during the exponential phase or to the appearance of a stationary-phase-specific activator. The proximity of the sin and inhA genes suggested that inhA expression may be regulated by SinR. The involvement of sinI and sinR genes in the regulation of B. thuringiensis inhA gene expression was investigated by comparing the production of ß-galactosidase in B. thuringiensis 407 [inhA'–lacZ] overexpressing the sinI or sinR gene. These two genes were cloned separately downstream from the xylose-inducible promoter in the high-copy-number plasmid pHT315 (see Methods). The plasmids pHT315xyl{Omega}sinI and pHT315xyl{Omega}sinR were introduced into B. thuringiensis 407 [inhA'–lacZ] by electroporation, and the cells were grown in HCT medium supplemented with xylose (20 mM). Overexpression of sinR delayed the inhA-directed ß-galactosidase synthesis, whereas ß-galactosidase expression started earlier in the B. thuringiensis strain overexpressing sinI than in the wild-type B. thuringiensis (Fig. 3a).



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Fig. 3. Expression of inhA'–lacZ transcriptional fusions in various B. thuringiensis genetic backgrounds. (a) Effect of sinI or sinR overexpression on inhA transcription. Expression of inhA'–lacZ fusion was determined during growth at 30 °C in HCT medium supplemented with xylose (20 mM). ß-Galactosidase activities were determined in B. thuringiensis 407 [inhA'–lacZ] ({square}), carrying pHT315xyl{Omega}sinI ({blacktriangleup}) or pHT315xyl{Omega}sinR ({triangleup}). (b) Expression of inhA in a B. thuringiensis spo0A null mutant. ß-Galactosidase activities were determined in the B. thuringiensis 407 wild-type strain ({blacksquare}) and in the B. thuringiensis 407 {Delta}spo0A null mutant ({circ}) carrying pHT304{Omega}inhA'–lacZ. The cells were grown at 30 °C in HCT medium.

 
Effect of spo0A null mutation on inhA expression
Spo0A is the key factor controlling the transcription of genes involved in the initial stages of sporulation. Expression of the inhA'–lacZ fusion was analysed in a B. thuringiensis {Delta}spo0A mutant strain. Wild-type 407 cells and the {Delta}spo0A mutant strain harbouring the pHT304{Omega}inhA'–lacZ plasmid were grown in HCT medium, and ß-galactosidase activities were assayed at various stages of growth (Fig. 3b). The spo0A mutation prevented the activation of the inhA transcription at the onset of the stationary phase, indicating that Spo0A is required for inhA expression. However, analysis of the inhA promoter region (Fig. 1a) did not reveal a putative 0A-box (TGTCGAA) (Hoch, 1993b ), thus suggesting that Spo0A may control inhA expression indirectly.

Expression of the inhA gene in B. subtilis is also dependent on Spo0A
We decided to study Spo0A-dependent regulation of the B. thuringiensis inhA gene in B. subtilis because numerous relevant mutant strains are available for B. subtilis but not for B. thuringiensis. ß-Galactosidase activity expressed by the B. subtilis 168 strains harbouring the pHT304{Omega}inhA'–lacZ plasmid was followed in cells growing in a sporulation-specific medium (SP). In the B. subtilis wild-type strain, the ß-galactosidase activity was low during vegetative growth and rose from about 200 to 1110 Miller units between t0·5 and t2 (Fig. 4a). The inhA-directed ß-galactosidase activity in B. subtilis was much lower than in B. thuringiensis (about 1100 Miller units in B. subtilis versus 14000 Miller units in B. thuringiensis at t2). ß-Galactosidase activity was also assayed in a B. subtilis {Delta}spo0A mutant strain carrying pHT304{Omega}inhA'–lacZ. As in B. thuringiensis, the inhA-directed ß-galactosidase expression was substantially reduced in the B. subtilis {Delta}spo0A mutant (Fig. 4a). Thus, the inhA expression is not strictly similar in B. thuringiensis and B. subtilis, suggesting a difference between the two bacteria. However, the profile of inhA expression at the onset of the stationary phase is comparable in the two Bacillus species, such that work in B. subtilis should be informative about the mechanisms of Spo0A-dependent inhA expression.



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Fig. 4. Expression of the inhA'–lacZ fusion in various B. subtilis genetic backgrounds. (a) inhA expression, measured as ß-galactosidase activity, in B. subtilis wild-type ({blacksquare}) and B. subtilis spo0A null mutant ({bullet}) carrying pHT304{Omega}inhA'–lacZ. (b) ß-Galactosidase activity of B. subtilis 168 wild-type ({blacksquare}), {Delta}sigE ({bullet}), and {Delta}sigF ({circ}) strains carrying pHT304{Omega}inhA'–lacZ. (c) ß-Galactosidase activity of B. subtilis 168 wild-type ({blacksquare}), {Delta}abrB ({blacktriangleup}), {Delta}spo0A ({bullet}), and {Delta}spo0A–{Delta}abrB ({triangleup}) strains carrying pHT304{Omega}inhA'–lacZ. The cells were grown at 30 °C in SP medium.

 
Effect of {sigma}E, {sigma}F and abrB mutations on inhA expression in B. subtilis
The Spo0A protein functions in a dual manner, acting as both a positive and a negative regulator. It functions as a positive regulator on expression of three stage II genes, spoIIA, spoIIE and spoIIG (Satola et al., 1991 ; Trach et al., 1991 ; York et al., 1992 ), and represses the abrB gene (Strauch et al., 1989 a). To determine how Spo0A regulates inhA transcription, the expression of the inhA'–lacZ fusion was assayed in various genetic backgrounds. Expression of spoIIAC and spoIIGB genes, encoding the sporulation-specific sigma factors {sigma}F and {sigma}E, respectively, requires the phosphorylated form of Spo0A (Bird et al., 1993 ; Trach et al., 1991 ). Thus, although a typical {sigma}A-dependent promoter was identified upstream of inhA, the effect of spoIIAC and spoIIGB mutations on inhA expression was tested. The absence of {sigma}E or {sigma}F did not significantly modify inhA expression (Fig. 4b), and therefore inhA expression is independent of these sporulation-specific {sigma} factors. This is consistent with inhA expression being {sigma}A-dependent.

The expression of the inhA'–lacZ fusion was also assayed in a {Delta}abrB genetic background. The B. subtilis {Delta}abrB mutant carrying pHT304{Omega}inhA'–lacZ displayed an overall ß-galactosidase activity similar to that of the wild-type B. subtilis strain (Fig. 4c). This suggests that abrB mutation does not affect inhA expression. However, the activation of the inhA'–lacZ expression occurred earlier in the {Delta}abrB mutant than in the wild-type strain (Fig. 4c), suggesting that AbrB might repress inhA transcription. As observed for the regulation of aprE gene expression, AbrB repression was clearly demonstrated in vivo by removing Spo0A, the repressor of AbrB (Smith, 1993 ; Strauch & Hoch, 1993 ). We tested whether an abrB mutation bypassed the spo0A dependence of inhA transcription. The determination of the ß-galactosidase production profile in the B. subtilis {Delta}spo0A–{Delta}abrB mutant carrying pHT304{Omega}inhA'–lacZ revealed that the inhibition of inhA'–lacZ expression by the spo0A mutation was indeed reversed by an abrB mutation (Fig. 4c). These results indicate that the Spo0A-dependent regulation of inhA expression is due to the AbrB repressor.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We report the cloning and analysis of the transcriptional regulation of the inhA gene from B. thuringiensis 407. The DNA fragment containing the inhA gene from strain 407 also contained two ORFs encoding polypeptides similar to the SinI and SinR regulators of B. subtilis. The InhA protease from B. thuringiensis 407 presents 92% identity with the InhA protein from B. thuringiensis var. alesti (Lövgren et al., 1990 ). However, the first 109 amino acids are absent from the published sequence of the B. thuringiensis var. alesti inhA gene. The identification of a putative signal peptide cleavage site between positions 31 and 32, and the alignment on the complete length of the amino acid sequence of InhA and PrtV protease from Vibrio cholerae, strongly suggest that the first 109 amino acids were erroneously omitted from the published sequence of the InhA protein from B. thuringiensis var. alesti.

Analysis of the expression of the transcriptional inhA'–lacZ fusion showed that inhA transcription was activated at the onset of sporulation in B. thuringiensis. To determine the mechanism involved in the genetic regulation of inhA, the inhA'–lacZ expression was measured in various genetic backgrounds in B. thuringiensis and B. subtilis.

The induction of inhA expression in B. thuringiensis was abolished by a spo0A mutation. Because of the absence of a typical 0A box (TGT/GCGAA) in the promoter region of the inhA gene, we tested for intermediate gene(s) involved in the Spo0A-dependent repression of inhA. This was done in B. subtilis, in which many spo genes and stationary-phase regulators have been characterized, and in which the corresponding mutants are available. As in B. thuringiensis, the inhA'–lacZ fusion expression was activated at the onset of the sporulation in B. subtilis and this activation was prevented in a spo0A null mutant. Here we show that inhA'–lacZ expression in B. subtilis was AbrB-dependent. The B. subtilis AbrB protein participates in the regulation of processes associated with the end of the exponential phase. AbrB plays three different roles (repressor, activator and preventer) and prevents expression of post-exponential-phase genes at inappropriate times (Strauch & Hoch, 1993 ). The repressive effect of abrB on inhA gene expression, like that on aprE expression, is clearly observed only in a {Delta}spo0A background, resulting in a high concentration of AbrB during the stationary phase. However, the lack of inhA expression during exponential growth in {Delta}abrB and {Delta}abrB–{Delta}spo0A backgrounds suggests that an additional regulator is required to repress inhA transcription during exponential growth or to activate it at the onset of the stationary phase. As discussed below, this additional regulator might be the SinR protein.

The binding of AbrB to the promoter region of various B. subtilis genes has been demonstrated by DNase I footprinting (Strauch et al., 1989 b). In each case, relatively long stretches were protected, including one or more crucial promoter elements (-35, -10 and +1). Biochemical studies suggest that AbrB binding involves multiple recognition sites and is a co-operative process. Nevertheless, examination of known AbrB-binding regions indicates that AbrB probably recognizes a three-dimensional DNA structure rather than a typical DNA sequence (Strauch et al., 1989b ; Xu & Strauch, 1996 ). The spo0E binding site for AbrB can be subdivided in three smaller recognition elements, each capable of directing AbrB binding (Strauch, 1996 ). All of these elements possess a conserved AT–7 bp–AAT motif which, by itself, is not sufficient for AbrB binding, but may contribute to recognition or binding stability. A significantly high frequency of (AT–7 bp–AAT) motifs is observed in most known AbrB-binding regions. Three adjacent (AT–7 bp–AAT) motifs are localized in the -79 to +1 region of the inhA promoter (Fig. 1a). Moreover, an upstream A+T-rich sequence is required for optimal AbrB binding (Robertson et al., 1989 ), and the inhA promoter region from -37 to -95 is 85% A+T. All these data support the hypothesis that inhA expression is directly regulated by AbrB. The putative abrB gene of B. thuringiensis has, however, not been cloned.

Overexpression of sinR delayed inhA expression in B. thuringiensis, whereas overexpression of sinI resulted in an early inhA expression (Fig. 3a). In B. subtilis, SinR activity is regulated post-translationally: the protein SinI binds SinR, thereby inhibiting its activity (Bai et al., 1993 ). Thus, early expression of inhA in B. thuringiensis overexpressing sinI may be due to the SinR-antagonist activity of SinI. There are two possibilities for the effect of SinR on inhA expression: that SinR directly interacts with the inhA promoter region and acts as a transcriptional repressor, and/or that SinR-dependent regulation of inhA involves an intermediate factor. In B. subtilis, SinR is a repressor of spo0A (Mandic-Mulec et al., 1995 ). Assuming a similar phenomenon in B. thuringiensis, overexpression of sinR would result in a decrease of inhA transcription (as observed in Fig. 3a) by delaying the repression of abrB by Spo0A~P, thus causing increased levels of AbrB in the cell.

InhA is not essential for growth or sporulation. However, the regulation of production of InhA, subtilisin or other exoenzymes may be much more important for the survival of B. thuringiensis or B. subtilis in natural environments than in laboratory conditions. A homologous InhA protein has recently been found in an active form on the exosporium of B. cereus (Charlton et al., 1999 ). This suggests that the presence of InhA on the spore surface may have functional significance. It would be interesting to address this question, and especially to define the role of InhA in the virulence of B. thuringiensis in insects.


   ACKNOWLEDGEMENTS
 
We thank Georges Rapoport and Tarek Msadek for their advice and constant interest throughout the work and Pascale Servant for critical reading of this paper. We thank Alex Edelman for English corrections. This work was supported by the Institut Pasteur, the Institut National de la Recherche Agronomique (AIP Microbiologie), the Centre National de la Recherche Scientifique and the Université Paris VII.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 10 January 2001; revised 7 March 2001; accepted 22 March 2001.