GENE REGULATION

An Extracytoplasmic-Function Sigma Factor Is Involved in a Pathway Controlling β-Exotoxin I Production in Bacillus thuringiensis subsp. thuringiensis Strain 407-1

Sylvain Espinasse, Michel Gohar, Didier Lereclus, Vincent Sanchis
DOI: 10.1128/JB.186.10.3108-3116.2004

ABSTRACT

β-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for β-exotoxin I production in B. thuringiensis subsp. thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1(Cry−)(Pig+), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1(Cry−)(Pig+) mutant. This suggests that in the wild type-407(Cry+) strain, β-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce β-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that β-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

Bacillus thuringiensis is a sporogenic, gram-positive, opportunistic insect pathogen that is extensively used in biological pest control (40). It produces parasporal crystal proteins (designated Cry proteins) (46). The genes encoding the Cry proteins are generally located on large conjugative plasmids that are larger than 45 kbp, although in some strains a chromosomal location of the crystal genes has also been reported (19). A significant proportion of strains also secrete one or several other insecticidal proteins (14, 29, 52) or a nonspecific heat-stable exotoxin known as β-exotoxin I (15). This nonproteinaceous toxin is a 701-Da adenine-nucleotide analogue that is thought to inhibit RNA polymerase by competition with ATP (47). It is particularly active against dipteran insects on ingestion but is also active against coleopteran, lepidopteran, aphid, and some nematode species (5). At sublethal doses, this toxin affects insect molting and pupation and may result in adults with developmental defects (8). Several toxicological studies have also reported that this molecule has cytotoxic effects on mammalian cells (4, 34). The World Health Organization has therefore banned β-exotoxin I for use in public health (54). To date, most studies on β-exotoxin I have focused on its mode of action and toxicology (10, 36, 47), and few studies have been done to unravel the regulatory mechanisms and genetic determinants involved in β-exotoxin I production. However, β-exotoxin I production has often been linked to the presence of plasmids of various sizes bearing cry genes (12). Several experiments have shown that the ability to secrete β-exotoxin I and the ability to produce crystals were transferred together to Bacillus cereus and B. thuringiensis recipient strains by conjugation (32). Conversely, strains that had lost the capacity to synthesize crystal toxins following loss of cry plasmids also became unable to secrete β-exotoxin I, although the parental strains had the capacity for high production of this compound (41).

We recently reported isolation of a B. thuringiensis mutant strain, 407-1(Cry−)(Pig+), obtained by chemical mutagenesis, which secreted considerable amounts of β-exotoxin I and of a soluble pigment (melanin) (13, 50). This strain is derived from a B. thuringiensis acrystalliferous mutant strain, designated 407(Cry−), that had lost the capacity to synthesize crystal toxins following loss of its curable cry plasmids. As a consequence, this strain also became unable to secrete β-exotoxin I, although the parental 407(Cry+) strain had the capacity for high-level production of this compound. We used strain 407-1(Cry−)(Pig+) in an attempt to isolate putative chromosomal genetic determinants involved in both pigment and β-exotoxin I expression. This work resulted in the identification of a genetic locus harboring two genes, berA and berB, encoding putative ABC transporters that were necessary for β-exotoxin I production (13). In this study, we describe an extracytoplasmic-function (ECF) (33, 38, 44) sigma factor gene, homologous to the sigW gene of Bacillus subtilis (24, 53), the expression of which is upregulated and necessary for β-exotoxin I production in the 407-1(Cry−)(Pig+) strain.

MATERIALS AND METHODS

Bacterial strains and plasmids.The bacterial strains and plasmids used in this study are described in Table 1. For plasmid preparation and cloning experiments, Escherichia coli K-12 strain TG1[Δ(lac-proAB) supE thi hdsD5 (F′ traD36 pro+ proB+ lacIq lacZΔM15)] and strain SCS110 [rpsL (Strr) thr leu endA thi-1 lacY gal4 galT ara tonA tsx dam dcm sup E44 Δ(lac-proAB) (F′ traD36 proAB lacIqZΔM15)] (Stratagene, La Jolla, Calif.) were used as hosts.

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TABLE 1.

Strains and plasmids

Growth conditions.B. thuringiensis subsp. thuringiensis strains 407 and 407-1 and their derivatives containing lacZ transcriptional fusions were cultured at 30°C, with shaking at 170 rpm, in Luria-Bertani (LB) medium or brain heart infusion (Difco) supplemented with selective antibiotics (erythromycin at 10 μg/ml, spectinomycin at 300 μg/ml, and kanamycin at 200 μg/ml). We monitored the growth of the culture in liquid medium by measuring the optical density at 600 nm. T0, the point corresponding to the end of (i.e., 0 h after the end of) exponential growth, was determined by regression for each culture growth curve. E. coli K-12 strains TG1 and SCS 110 were cultured at 37°C in LB medium supplemented with the antibiotics ampicillin (100 μg/ml), erythromycin (5 μg/ml), and kanamycin (20 μg/ml).

Bioassays of insecticidal activity.We used a free-ingestion technique to assess the toxicity of bacterial culture supernatant preparations to Spodoptera littoralis (Lepidoptera). The strains were cultured on LB agar plates at 30°C. A single colony was then used to inoculate 100 ml of LB, which was then incubated for 24 h at 30°C with shaking. Cells were harvested from the resulting cultures at midsporulation (before cell lysis) by centrifugation at 14,000 × g for 10 min at 4°C, and the supernatants were filtered twice through Nalgene filter units with 0.2-μm pores. The resulting extracts were stored at −20°C until they were used for toxicity assays and β-exotoxin I determination. S. littoralis larvae were fed an artificial diet dispensed into 50-well plastic plates (well area, 1.65 cm2). Supernatant (25 μl) was applied uniformly over the food surface and allowed to dry. We placed one neonatal larva on each of 35 wells and incubated the plate for 10 days at 25°C with a photoperiod (light/dark) of 16:8 h and 70% relative humidity. Mortality was recorded on days 3, 6, and 10. Under these conditions, the concentration of toxin required to kill 50% of the S. littoralis neonates, using technical-grade β-exotoxin I, was 30 μg/ml. Typically, the larvae were impaired in molting and developed into aberrant first instars which were white and puffy.

Detection and quantification of β-exotoxin I.β-Exotoxin I was extracted from the culture supernatant by solvent extraction and quantified by high-pressure liquid chromatography (HPLC) (17). Briefly, for solvent extraction, acetone was added, to a final concentration of 90%, to the exotoxin in 0.2 ml of culture supernatant and the mixture was centrifuged. The pellet was solubilized in 0.2 ml of double-distilled water. Acetonitrile was added to a final concentration of 40%, and the mixture was centrifuged. The pellet was discarded, and the acetonitrile concentration of the supernatant was brought up to 90%. The precipitate was collected by centrifugation, and the pellet was solubilized in 100 μl of 50 mM potassium phosphate buffer (pH 2.5). For HPLC, we injected 25 μl of the sample into a Lichrospher (Merck) C18 end-capped 4- by 250-mm column. A gradient of 5 to 15% methanol in 50 mM potassium phosphate buffer (pH 2.5) was developed over 10 min. The flow rate was 1 ml/min, and UV absorption was monitored at 260 nm. β-Exotoxin I was eluted at 5.5 min. The detection limit of this method was 2 μg/ml. I. Thiery (Laboratoire des Bactéries Entomopathogènes, Institut Pasteur, Paris, France) kindly provided a standard sample (70% purity) of β-exotoxin I.

Plasmid construction.The plasmids used in this study are listed in Table 1, and the oligonucleotides used are listed in Table 2. Subregions of the DNA fragment shown in Fig. 1 were amplified by PCR from chromosomal 407(Cry−) DNA and inserted into pHT304-18lacZ (1), between the PstI and BamHI sites, to generate transcriptional fusions with the lacZ reporter gene.

FIG. 1.
FIG. 1.

Analysis of the DNA region located downstream of the berB gene in B. thuringiensis. A schematic representation of the studied region of B. thuringiensis 407(Cry−) deduced from its nucleotide sequence and delineation of the sigW promoter region by using different lacZ fusions are shown. The various ORFs are boxed, with their sizes (in base pairs) and directions of transcription indicated. The promoter region (Pw) of sigW and the potential stem-loops are also indicated. The arrows indicate the various fragments cloned into pHT304-18Z to generate transcriptional fusions (constructs pHT304-18ΩPw1-lacZ to pHT304-18ΩPw7-lacZ) for determination of promoter activity in B. thuringiensis. The nucleotide positions at the ends of the cloned fragments are given with reference to the translational start codon of sigW (position +1). ND, not determined.

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TABLE 2.

Oligonucleotides used in this study

The primers used for the construction of the different plasmids used for the delimitation of the DNA region containing the sigW promoter (constructs pHT304-18ΩPw1-lacZ to pHT304-18ΩPw7-lacZ) and pHT304-18ΩsigWecfXecfY-lacZ, with their added restriction sites, are listed in Table 2. B. thuringiensis strains were transformed by electroporation. The sigW, ecfX, and ecfY genes were overexpressed by inserting the fragments of the sequence corresponding to sigW (primers RBS-XbaI/BamHI-orf3), sigW-ecfX (primers RBS-XbaI/BamHI-orf4), and sigW-ecfX-ecfY (primers RBS-XbaI/BamHI-orf5-entier) into the reconstituted multicopy expression plasmid pHT16-18Pxyl. The pHT304-18lacZ plasmid, used for the fusions, and the pHT16-18Pxyl plasmid possess different origins of replication (31). The xylose-induced Pxyl promoter includes the promoter of xylA from B. subtilis and a functional copy of the xylR regulator, which represses this promoter in the absence of xylose (16, 28). Pxyl was extracted from PHTS2xyl as previously described (18) and inserted into pHT16-18 between the HindIII and BamHI sites. Promoter activity was induced by adding 20 mM xylose to the culture medium at 30°C.

Construction of sigW, ecfX, and ecfY mutants.We disrupted the sigW, ecfX, and ecfY genes in B. thuringiensis strains 407(Cry+) and 407-1(Cry−)(Pig+) by amplifying the DNA regions 5′ and 3′ to each coding sequence by PCR, using oligonucleotides including appropriate restriction sites (Table 2). The PCR fragments were hydrolyzed with BamHI-XbaI and EcoRI-EagI and ligated with a 1.6-kb XbaI-EcoRI DNA fragment carrying the aphA3 gene of Enterococcus faecalis (Kmr cassette) (37). The disrupted gene copies were inserted into a pRN5101 derivative, which was constructed by A. Gruss (INRA, Jouy en Josas, France) by inserting pE194ts (48) into the ClaI site of pBR322. The replication of this plasmid is heat sensitive in gram-positive hosts. The chromosomal copies of sigW, ecfX, and ecfY were replaced by disrupted copies of the corresponding genes by double crossing over, as described by Bravo et al. (6). The recombinant strains were selected at a nonpermissive temperature on LB plates supplemented with kanamycin. Strains with disrupted genes were kanamycin resistant and erythromycin sensitive. Each gene disruption was checked by PCR, using relevant primers binding to the chromosomal sequence and k1 and k2 primers, which bind within the internal aphA3 sequence.

β-Galactosidase assays.B. thuringiensis cells containing lacZ transcriptional fusions were cultured in LB medium at 30°C, and β-galactosidase assays were performed as previously described (39). Specific activities are expressed in units of β-galactosidase per milligram of protein (Miller units).

Primer extension assays.RNA was prepared from a single culture of B. thuringiensis 407(Cry−)(Pig+), sampled at times T0, T2, and T4 (LB medium, 30°C, 170 rpm), as previously described (2). For each sample, total RNA (100 μg) was incubated with 2 pmol of 32P-end-labeled reverse primer Trev.ECF (Table 2). The resulting precipitate was collected by centrifugation, and the pellet was resuspended in 40 μl of hybridization buffer (60 mM NaCl, 50 mM Tris-HCl [pH 8.0], 10 mM dithiothreitol) heated to 95°C for 3 min and cooled slowly to 40°C over 30 min. We then added 60 μl of extension solution (60 mM NaCl, 50 mM Tris-HCl [pH 8.0], 13 mM dithiothreitol, 10 mM MgCl2, 1 mM deoxynucleoside triphosphates, 20 U of avian myeloblastosis virus reverse transcriptase [Gibco-BRL]), and the mixture was incubated at 37°C for 30 min (24). Nucleic acids were purified and concentrated on spin columns by using a PCR purification kit (Qiagen), resuspended in 20 μl of water supplemented with 0.1 μg of DNase-free RNase, and incubated at room temperature for 30 min. The nucleic acids were separated by electrophoresis in an 8 M urea-6% polyacrylamide gel, with a sequencing reaction mixture from primer Trev.ECF loaded alongside the other lanes for comparison. Before loading, the samples were mixed with sequencing loading buffer (5 μl) and heated for 3 min at 95°C. The bands on the gel were visualized by autoradiography.

Nucleotide sequence accession number.The nucleotide sequence of the region harboring sigW, ecfX, and ecfY has been deposited in GenBank under accession no. AF499614.

RESULTS

Nucleotide and protein sequence analyses of a genetic locus harboring an ECF-type sigma factor.In a previous study, we performed a mini-Tn10 transposition experiment with strain 407-1(Cry−)(Pig+) to identify genes involved in β-exotoxin I production. This approach made it possible to identify two overlapping genes, berA and berB, encoding an ABC transporter necessary for β-exotoxin I production (13). Analysis of the nucleotide sequence adjacent to the insertion site revealed the presence, downstream from the berB gene, of a putative polycistronic operon composed of three open reading frames (ORFs) separated from berB by a hairpin structure and 600 bp of noncoding sequence (Fig. 1). The first ORF, ORF1, encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase ECF-type sigma factors from Bacillus anthracis strain Ames (45) and B. cereus strain ATCC 14579 (26), respectively (accession numbers NP_845915 and NP_833321, respectively). It was also 50, 30, and 26% identical to SigW from Clostridium perfringens (accession number BAB80422) and SigW (23) and SigX (7, 22) from B. subtilis, respectively. SigW and SigX from B. subtilis are alternative sigma factors belonging to the ECF-type subfamily of RNA polymerase sigma factors (38, 44). The functional domains involved in RNA polymerase core binding and in recognition of the −10 and −35 regions are well conserved in the predicted ORF1 polypeptide (Fig. 2). Blast searches for the two other ORFs (ORF2 and ORF3), encoding putative proteins of 332 and 345 amino acids in length, revealed no substantial similarity in amino acid sequence to proteins of known function present in the databases. However, the ORF2 protein displayed 96% amino acid identity to a putative ECF-type sigma factor negative effector from B. cereus ATCC 14579 (accession number NP_833320) and 87% identity to an hypothetical protein from B. anthracis strain Ames (accession number NP_845914). ORF2 also displayed 50% amino acid identity to a putative protein from C. perfringens (accession number BAB804223) encoded by a gene located downstream from the C. perfringens sigW gene. The ORF2 protein possesses a putative membrane-spanning domain between residues 50 and 100, which is also found in Rsi-X (YpuN) (7), the anti-sigma factor for SigX of B. subtilis. The ORF3 protein is 95 and 91% identical to two putative antiterminators from B. anthracis Ames and B. cereus ATCC 14579 (accession numbers NP_883319 and NP_845913, respectively). It also displays 43% identity to a putative transcriptional regulator from Listeria monocytogenes (accession number CAD00596) that is similar to the B. subtilis membrane-bound protein LytR, the production of which is also under the control of SigX in B. subtilis (22, 23). We called these three putative genes sigW (like the ECF sigma factor of B. subtilis), ecfX, and ecfY, respectively.

FIG. 2.
FIG. 2.

Amino acid sequence comparison between the putative ECF sigma factors of B. thuringiensis (Bt), C. perfringens (Cp) (putative SigW), B. subtilis (Bs) (SigW), and E. coli (Ec) (SigE). The alignments were designed to optimize identity; similar amino acids are boxed in black. The resulting identity scores are as follows: C. perfringens, 50%; B. subtilis, 28%; and E. coli, 26%. Domains, numbered 2.1, 2.2, 2.3, 2.4, 4.1, and 4.2, that are involved in RNA polymerase core binding and in recognition of the −10 and −35 regions have been described by Lonetto et al. (33) and are conserved among ECF sigma factors.

Gene disruption experiments reveal that the sigW and ecfX genes are necessary for β-exotoxin I production in strain 407-1(Cry−)(Pig+).ECF sigma factor homologues have been identified as potential regulators of small-molecule synthesis and export in various situations. We generated chromosomal mutants of sigW, ecfX, and ecfY for investigation of the role of these genes in β-exotoxin I production in strains 407(Cry+) and 407-1(Cry−)(Pig+). The genes were disrupted by insertion of the aphA3 gene (see Materials and Methods) in the reverse orientation. In each case, part of the internal gene sequence was removed. In strain 407-1(Cry−)(Pig+), replacement of the sigW and ecfX genes by their disrupted copies resulted in nontoxic mutants, the culture filtrates of which yielded 4.5 and 1.7 μg of β-exotoxin I per ml, respectively, as determined by HPLC. This is a very small amount of β-exotoxin I given that strain 407-1(Cry−)(Pig+) produced 172 μg/ml (Table 3). By contrast, strain 407-1(Cry−)(Pig+)ΔecfY displayed no impairment of β-exotoxin I production. The concentration of 50 μg of β-exotoxin I per ml found in the supernatant of strain 407(Cry+)ΔsigW was not significantly different from that found in the wild-type strain, 407(Cry+).

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TABLE 3.

Insecticidal activities and β-exotoxin I concentrations in culture supernatants of various B. thuringiensis strain 407 derivatives

Expression of sigW in the 407 variants.A transcriptional fusion between a DNA fragment including the coding sequences of sigW, ecfX, and ecfY and the lacZ reporter gene (pHT304-18ΩsigWecfXecfY-lacZ construct) was assayed for β-galactosidase activity. No promoter activity was detected within the sigW-ecfX-ecfY cluster (data not shown), suggesting that this cluster may constitute an operon (Fig. 1). A transcriptional fusion between the 723-bp DNA sequence located upstream from sigW and the lacZ gene was then constructed from pHT304-18Z. This fusion (Pw1 construct [Fig. 1]), which extended from nucleotide position −585 to +138 (with reference to the translational start codon of sigW as position +1), was introduced into strains 407-1(Cry−)(Pig+), 407-1(Cry−)(Pig+)ΔsigW, 407(Cry−), and 407(Cry+). β-Galactosidase activity was assessed during both the exponential and stationary phases of bacterial growth. In strain 407-1(Cry−)(Pig+) containing this fusion, the level of β-galactosidase activity increased from 50 Miller units at T2 to 300 Miller units at T8, indicating that, in this strain, a promoter (Pw) was active during the stationary phase. In strains 407(Cry−) and 407(Cry+), this promoter fusion expressed the lacZ reporter gene to give background levels of activity only (β-galactosidase activities of 25 and 70 Miller units at T2 and T8, respectively), indicating absent or weak transcription of sigW in these strains. The expression of lacZ from Pw1 was also found to be reduced to background levels in strain 407-1(Cry−)(Pig+)ΔsigW. Thus, Pw activity in the 407-1(Cry−)(Pig+) genetic background requires expression of the sigW operon and suggests a positive autoregulation similar to that observed for most ECF sigma factor genes.

Delimitation of the DNA region containing the sigW promoter and mapping of the 5′ ends of sigW transcripts.The Pw1 DNA fragment contains the promoter region of sigW. To determine the minimal region required for full expression of sigW, the region extending from nucleotide position −585 to +1675 (with reference to the translational start codon of sigW) was subjected to deletion analysis. A series of deletion fragments were cloned upstream of the lacZ reporter gene in plasmid pHT304-18Z (constructs pHT304-18ΩPw1-lacZ to pHT304-18ΩPw7-lacZ) (Fig. 1). These plasmids were introduced into B. thuringiensis strains 407-1(Cry−)(Pig+) and 407-1(Cry−)(Pig+)ΔsigW, and the production of β-galactosidase was measured. The levels of β-galactosidase activity expressed at T6 (6 h after the onset of stationary phase) by the strains harboring the different plasmids are shown in Fig. 1. These data indicate that the DNA sequence that contains the Pw promoter can be reduced to a shorter segment covering the 154 bp upstream from the GTG start codon (Pw5 construct). The levels of β-galactosidase activity expressed from Pw2 were equivalent to those obtained with Pw5, indicating that the potential stem-and-loop structure does not seem to play a specific role in the expression of sigW. A fusion, extending from nucleotide position −199 to +1676 (Pw7 construct), between PwsigWecfX and lacZ resulted in a large decrease in β-galactosidase production, indicating that EcfX exerts a negative control in the expression of sigW (compare Pw7 with Pw2 and Pw5). No promoter activity was detected in the region located upstream from the potential stem and loop (Pw3 and Pw4 constructs) or between the Pw promoter region and ecfX (Pw6 construct).

The 5′ end of the of sigW mRNA was then identified, using 100 μg of RNA template prepared from 407-1(Cry−)(Pig+) cells grown at 30°C in LB medium and sampled at T0, T2, and T4. The DNA fragments obtained from transcripts were detected by reverse transcription (Fig. 3A). Primer extension revealed that sigW transcripts were present at T2 and T4 but not at T0. This is consistent with the results of the Pw-lacZ fusion experiment, confirming that transcription starts after the onset of the stationary phase. One RNA 5′ end was identified as corresponding to a sequence 51 bases upstream from the sigW start codon (Fig. 3B). The GAAACCTT and CGTCTA sequences of the region located 35 and 10 bp upstream from the transcription start site were similar to those described for Pw and Px, the respective promoters of sigW and sigX from B. subtilis (22). Alignment and comparison of the sequences of the B. thuringiensis and B. subtilis sigW promoters predicted a consensus (−35)G(A)AACCTTN14(−10)CGT(N)TA for these two autoregulated sigma factors (Fig. 3C).

FIG. 3.
FIG. 3.

Analysis of the Pw promoter region. (A) Determination of the transcriptional start site of sigW by primer extension assay. Total RNA was extracted from B. thuringiensis 407-1(Cry−)(Pig+) at the onset of the stationary phase (T0) and at T2 and T4. RNA was subjected to primer extension with the oligonucleotide Trev.ECF (Table 2), extending from position +55 to +78, with reference to the translational start codon of sigW (position +1), which was also used for sequencing reactions shown in base order A, C, G, and T. (B) Nucleotide sequence of the Pw region. The identified putative −10 and −35 boxes are underlined. The transcriptional start site and the GTG start codon are shown in boldface. (C) Nucleotide sequence comparison between the Pw promoters of the B. thuringiensis (Bt) and B. subtilis (Bs) sigW genes.

Effects of ecfX and ecfY expression on Pw transcription.We investigated the role of EcfX and EcfY in the expression of sigW by analyzing the expression of the Pw5-lacZ fusion (pHT304-18ΩPw5-lacZ construct) in strain 407-1(Cry−)(Pig+)sigW::aphA3 carrying a multicopy plasmid (pHT16-18) expressing sigW, sigW-ecfX, or sigW-ecfX-ecfY under the control of the xylose-inducible promoter Pxyl. In the presence of 20 mM xylose, we observed a high level of lacZ transcription in strain 407-1(Cry−)(Pig+)sigW::aphA3(pHT304-18ΩPw5-lacZ) transformed with pHT16-18ΩPxyl-sigW (Fig. 4), which suggests that Pw is autoregulated. When the strain was transformed with pHT16-18ΩPxyl-sigWecfX, the Pw promoter activity was 1/10 of that observed in the strain expressing only sigW. Finally, transformation with pHT16-18ΩPxyl-sigWecfXecfY fully repressed lacZ transcription.

FIG. 4.
FIG. 4.

sigW expression is autoregulated and negatively controlled by the products of expression of ecfX and ecfY. Expression of β-galactosidase from a Pw-lacZ fusion (carried by pHT304-18ΩPw5-lacZ) in strain 407-1(Cry−)(Pig+)sigW::aphAIII transformed with the different pHT16-18Pxyl constructs used to overexpress the sigW-ecfX-ecfY region is shown. •, sigW alone was overexpressed (pHT16-18ΩPxyl-sigW construct); ⧫, sigW and ecfX were overexpressed (pHT16-18ΩPxyl-sigW-ecfX construct); ▪, sigW, ecfX, and ecfY were overexpressed (pHT16-18ΩPxyl-sigW-ecfX-ecfY construct). The strains were cultured in LB medium supplemented with 20 mM xylose at 30°C.

DISCUSSION

We report here the identification of a genetic locus, encoding proteins, involved in the regulation of the synthesis of β-exotoxin I in B. thuringiensis strain 407-1(Cry−)(Pig+). Analysis of the DNA sequence downstream from the berAB genes, which are essential for β-exotoxin I production (13), revealed the presence of a gene cluster, designated sigW-ecfX-ecfY, that was associated with the high levels of β-exotoxin I production by strain 407-1(Cry−)(Pig+). The deduced amino acid sequence of the protein encoded by sigW displayed significant similarity to those of sigma factors of the subfamily of σ70-type proteins known as ECF proteins, which control the response to the environment by regulating genes encoding adaptive proteins (38, 44). ECF sigma factors are more variable than other sigma factors but have conserved domains, numbered 2.1, 2.2, 2.3, 2.4, 4.1, and 4.2 by Lonetto et al. (33) (Fig. 2), that are involved in RNA polymerase core binding and in recognition of the −10 and −35 regions. Amino acid sequence comparisons demonstrated that the putative SigW protein that was identified displayed relevant similarity to SigW of C. perfringens and to SigW and SigX of B. subtilis. These three genes have also very similar −10 boxes (CGTNTA), which are involved in the narrow specificity of the promoters for their cognate ECF sigma factors (42). This suggests a higher structural similarity between the target promoters of the genes controlled by sigW of B. thuringiensis and those of the sigW and sigX controlled genes of B. subtilis than between those of other ECF sigma factors. By contrast, the −35 box GAACCTT is general to most ECF members, as it binds region 4, which is conserved among ECF sigma proteins (33). For instance, rpoEP2 of sigE in E. coli has a GAACTT sequence (43), Pa of algU in Pseudomonas aeruginosa has the sequence GAACT (21), and PfecI of fecI in E. coli has GAAAC (11). Many putative ECF sigma factor genes with similarity to the sigW gene of B. subtilis have recently been identified in the genome sequences of gram-positive bacteria, but little has been done to unravel the functions of these genes. The function of sigW in B. subtilis is not clear. However, there is some evidence that it is involved in the transcription of various genes with roles in detoxification or the production of antimicrobial compounds and resistance to oxidative stress in response to diverse extracellular stimuli (24, 44). For example, SigW controls the expression of genes encoding various putative ABC transporters (35), a peroxidase, and an epoxide hydrolase and of fosB, a gene that confers resistance to the antibiotic fosfomycin (9). Moreover, several mutations upregulating sigW expression have been identified in various genes involved in detoxification and multidrug efflux (49).

Transcriptional lacZ fusion experiments and analysis of sigW transcripts suggested that sigW was autoregulated (Fig. 1) and that its expression in strain 407-1(Cry−)(Pig+) was activated during the stationary phase (transcripts were present at T2 and T4 but not at T0). Disruption of sigW in strain 407-1(Cry−)(Pig+) resulted in a nontoxic mutant that produced low levels of β-exotoxin I, whereas its deletion in the wild-type strain did not impair β-exotoxin I production. This suggests that in strain 407(Cry+), β-exotoxin I expression is probably induced by the expression of regulatory plasmid-borne genes that do not require SigW, whereas this is not the case in strain 407-1(Cry−)(Pig+). Moreover, the fact that sigW was activated at the onset of stationary phase in the mutant strain but not in the wild-type background could indicate that, under certain environmental conditions, sigW may facilitate the production of β-exotoxin I in the absence of δ-endotoxin plasmids. However, the nature of the mutation that activates sigW in strain 407-1(Cry−)(Pig+) and the stimulus for its expression in B. thuringiensis under natural conditions remain unknown.

EcfX, encoded by the gene downstream from sigW, significantly repressed the expression of sigW when both genes were overtranscribed (Fig. 1 and 4). Negative control in ECF sigma factors generally implies the existence of a specific cognate anti-sigma factor, the gene for which is usually cotranscribed with its corresponding sigma factor gene. In general, anti-sigma factors share very little sequence identity (20), but the proteins they encode are known to possess the hydrophobic domains required for insertion into the membrane. The EcfX protein displays a hydrophobic domain between residues 50 and 100, which may include a membrane domain as described for Rsi-X, the anti-sigma factor for SigX (25). Therefore, EcfX may be the anti-sigma factor for SigW in B. thuringiensis. However, disruption of the ecfX gene in strain 407-1(Cry−)(Pig+) resulted in a toxin-deficient mutant (Table 3). This indicates that expression of SigW alone is not sufficient to induce β-exotoxin I synthesis and that EcfX is required for β-exotoxin I production. The reason for the absence of β-exotoxin I production in this mutant strain is not known. It could be that EcfX is also required for the activation of sigW or that it protects SigW from degradation. It is also possible that SigW is not produced in an active form and that its sequestration by its putative anti-sigma factor is required to activate it upon the perception of an external signal. Finally, EcfY overexpression resulted in complete repression of the sigW-ecfX-ecfY operon, indicating that this protein, which shares some similarity with the B. subtilis membrane-bound transcriptional attenuator LytR, also exerts a negative control on sigW expression. Additional regulators frequently help to stabilize the sigma/anti-sigma factor complex (25, 38).

The presence of these genes in the genomes of B. cereus and B. anthracis, two human pathogens, could suggest that these two species also have the potential capacity to produce β-exotoxin I. Despite a high level of chromosomal similarity with B. thuringiensis, the three species occupy different ecological niches and have different lifestyles (27). Differential gene expression (chromosomal or plasmid) in response to host-specific sensing could explain the different physiological and virulence properties found between the various strains. In the case of B. thuringiensis, which is generally regarded as an insect pathogen, its ability to produce high levels of β-exotoxin I during stationary phase can make a significant contribution towards determining the insecticidal effects of individual strains. This could explain why genetic determinants involved in β-exotoxin I production are present on the same plasmids as certain cry genes (12). These plasmid-borne elements are more likely to be regulatory genes, whereas the genes responsible for β-exotoxin I biosynthesis are probably chromosomal. Therefore, in a large number of B. thuringiensis strains, it seems that β-exotoxin I synthesis can be activated through a nonchromosomal regulatory pathway which can confer a particular evolutionary advantage on the host strain. Genes located on the chromosome can be controlled by plasmid regulators. For example, in B. anthracis, PagR encoded by a plasmid gene, controls the transcription of the chromosomal sap gene, encoding an S-layer protein (37). For the other species belonging to the B. cereus group, for which expression of β-exotoxin I does not seem to rely on plasmid genetic determinants, the biological and ecological role and the conditions suitable for expression of this molecule remain to be determined.

In summary, we found that the activation of sigW expression was required for β-exotoxin I production in strain 407-1(Cry−)(Pig+). In the wild-type 407(Cry+) strain, sigW was not activated at the onset of stationary phase, and β-exotoxin I production depended on genetic determinants located on a plasmid. These results are consistent with previous observations indicating that β-exotoxin I production is differentially activated in the B. thuringiensis 407-1(Cry−)(Pig+) and 407(Cry+) strains (13). The sigW gene in strain 407-1(Cry−)(Pig+) is autoregulated, and the ecfX and ecfY genes, located downstream from sigW, negatively control its expression. The involvement of an ECF sigma factor in β-exotoxin I production, whether this involvement is direct or indirect, suggests that β-exotoxin I production can be activated in response to particular environmental conditions. Future studies will aim to characterize the genes whose transcription involves the product of sigW in B. thuringiensis strain 407-1(Cry−)(Pig+) and to investigate their involvement in β-exotoxin I production. It would also be interesting to establish whether this phenomenon occurs in other subspecies and strains of B. thuringiensis that do not produce β-exotoxin I.

ACKNOWLEDGMENTS

The B. thuringiensis mutant strain 407-1(Cry−)(Pig+) was kindly provided by O. Arantes (Universidade Estadual de Londrina, Londrina, Brazil). This work was supported by the Institut Pasteur and the Institut National de la Recherche Agronomique (INRA). Funds for sequencing were supplied by the European Community (EC contract Bio-CT96-0655). Sylvain Espinasse was supported by a grant from Aventis Crop-Science, Ghent, Belgium.

FOOTNOTES

    • Received 23 October 2003.
    • Accepted 23 January 2004.

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