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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 sinIsinR 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 proteinprotein 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
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.
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METHODS |
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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
-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
inhA'lacZ.
The plasmids pHT315pxylsinI and pHT315
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
sinI and pHT315xyl
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 HindIIIEcoRI fragment and as a 1 kb EcoRIBamH1 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 pHT304inhA'lacZ.
B. subtilis 168 containing pHT304inhA'lacZ was transformed with chromosomal DNA purified from
sigE,
sigF,
spo0A,
abrB and
spo0A
abrB B. subtilis mutants (laboratory stock) (Msadek et al., 1998
). The recombinant strains were selected for erythromycin and kanamycin resistance (
sigE,
sigF and
spo0A mutants containing pHT304
inhA'lacZ), erythromycin and chloramphenicol resistance (
abrB mutant containing pHT304
inhA'lacZ), or erythromycin, kanamycin and chloramphenicol resistance (
spo0A
abrB mutant containing pHT304
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
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 (212300 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.
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RESULTS |
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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
A promoter consensus (TTGACA 1718N 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 (pHT304inhA'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
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
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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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 pHT304inhA'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
spo0A mutant strain carrying pHT304
inhA'lacZ. As in B. thuringiensis, the inhA-directed ß-galactosidase expression was substantially reduced in the B. subtilis
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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The expression of the inhA'lacZ fusion was also assayed in a abrB genetic background. The B. subtilis
abrB mutant carrying pHT304
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
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
spo0A
abrB mutant carrying pHT304
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.
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DISCUSSION |
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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
spo0A background, resulting in a high concentration of AbrB during the stationary phase. However, the lack of inhA expression during exponential growth in
abrB and
abrB
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 AT7 bpAAT motif which, by itself, is not sufficient for AbrB binding, but may contribute to recognition or binding stability. A significantly high frequency of (AT7 bpAAT) motifs is observed in most known AbrB-binding regions. Three adjacent (AT7 bpAAT) 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.
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ACKNOWLEDGEMENTS |
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Received 10 January 2001;
revised 7 March 2001;
accepted 22 March 2001.