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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2013 Jun;195(12):2887–2897. doi: 10.1128/JB.00112-13

Transcriptional Regulation and Characteristics of a Novel N-Acetylmuramoyl-l-Alanine Amidase Gene Involved in Bacillus thuringiensis Mother Cell Lysis

Jingni Yang a,b, Qi Peng b, Zhen Chen b, Chao Deng b, Changlong Shu b, Jie Zhang a,b,✉, Dafang Huang c, Fuping Song b,✉
PMCID: PMC3697243  PMID: 23603740

Abstract

In Bacillus thuringiensis, a novel N-acetylmuramoyl-l-alanine amidase gene (named cwlB) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA) formed one transcriptional unit. 5′ rapid amplification of cDNA ends (5′-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, PcwlA, which is located upstream from the cwlA gene and that the transcription start site is a single 5′-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of PcwlA was controlled by σK. Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis.

INTRODUCTION

Bacillus thuringiensis is a Gram-positive, spore-forming bacterium belonging to the Bacillus cereus group that produces parasporal crystal proteins. These parasporal crystal proteins are encoded by the cry or cyt genes and possess highly specialized insecticidal activity against a large number of insect species (1). Because of the insecticidal properties of the crystal proteins, B. thuringiensis has been commercially used for many years as a pesticide in biocontrol applications (2). However, one of the problems encountered after applying B. thuringiensis in the field is a lack of stability of the crystals on plant leaves due to inactivation by environmental factors, such as UV light (3, 4). A previous study showed that the cell envelope was sufficient to protect against B. thuringiensis crystal degradation under UV sunlight and had no significant effect on insecticidal activity. The genetic construction with σK disruption did not sporulate, still produced toxins, and did not result in lysis of the mother cell (5). However, disruption of the σK factor can decrease both the transcription of some cry genes during late sporulation and the biomass (6–8). Thus, developing an alternative method that will block mother cell lysis but have no effect on sporulation and Cry protein production is important.

Peptidoglycan hydrolases, some of which are known as autolysins, play an essential role in mother cell lysis of Bacillus strains; these proteins form a vast and highly diverse group of enzymes capable of cleaving bonds in polymeric peptidoglycan (sacculi) and/or its soluble fragments (9, 10). Four different families have been defined according to the chemical bond cleaved in the peptidoglycan molecule: N-acetylmuramidase, N-acetylglucosaminidase, N-acetylmuramyl-l-alanine amidase, and endopeptidase (11). These enzymes participate in bacterial cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division, and autolysis (12–15). Three autolysins were reported to be involved in the mother cell lysis of Bacillus subtilis. Two autolysins, CwlB and CwlC, are present in large amounts at the time of mother cell lysis (16–18). A third, sporulation-specific amidase (CwlH), has also been shown to be required for mother cell lysis and acts in a compensatory manner with CwlC. Single inactivation of cwlB, cwlC, or cwlH has not been shown to affect mother cell lysis. However, mother cell lysis was found to be blocked in a mutant that had multiple inactivated genes (19). In the B. cereus group, the cell wall hydrolases that are responsible for mother cell lysis still remain unknown.

The roles of several peptidoglycan hydrolases from the B. cereus group have been studied. The spore-lytic enzymes cortical-fragment-lytic enzyme (CFLE; sleL gene) and spore cortex-lytic enzyme (SCLE; sleB gene) were found in the exudate of germinated B. cereus IFO 13597 spores. These enzymes differ from each other in the morphology of the substrates that they recognize; CFLE requires disrupted spore peptidoglycan for its activity, and SCLE preferentially hydrolyzes intact spore peptidoglycan (20, 21). The potential cell wall peptidase CwpFM from B. cereus ATCC 14579 was shown to be involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence (22). These peptidoglycan hydrolases have not been reported to be involved in mother cell lysis.

In this study, two open reading frames (ORFs) (bt2493 and bt2494, both encoding putative peptidoglycan hydrolases) were identified in the B. thuringiensis HD73 genome, and we named these two genes cwlA and cwlB, respectively. We also confirmed that cwlB is a novel cell wall hydrolase gene expressed during B. thuringiensis sporulation, characterized the activity of the enzyme it encodes, and determined the localization, role in mother cell lysis, and transcriptional pattern of the enzyme. Our findings will better elucidate the mechanism of mother cell lysis in B. thuringiensis and provide new insight into the development of biocontrol agents with better persistence.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli TG1 was used as the host for the cloning experiments, while SCS110 was used to generate unmethylated plasmid DNA for B. thuringiensis transformations (23, 24). B. thuringiensis HD73 was used as the recipient strain to monitor promoter activity and clone the target gene (25, 26).

Table 1.

Strains and plasmids used in this study

Strain or plasmid Characteristics Reference or source
E. coli strains
    TG1 Δ(lac-proAB) supE thi hsd-5 (F′ traD36 proA+ proB+ lacIq lacZΔM15) 23
    SCS110 Δ(lac-proAB) rpsL(Strr) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44 (F′ traD36 proAB lacIq lacZΔM15) 24
    BL21(DE3) F− dcm ompT hsdS(rB− mB−) gal λ(DE3) 33
    BL21(pETcwlB) BL21(DE3) with pETcwlB plasmid This study
    BL21(pGEXgerE) BL21(DE3) with pGEXgerE plasmid This study
B. thuringiensis subsp. kurstaki strains
    HD73 Contains cry1Ac gene 25, 26
    HD(ΔsigK) HD73 mutant, ΔsigK 8
    HD(ΔgerE) HD73 mutant, ΔgerE This study
    HD(ΔcwlA) HD73 mutant, ΔcwlA This study
    HD(ΔcwlB) HD73 mutant, ΔcwlB This study
    HD(ΔcwlAΔcwlB) HD73 mutant, ΔcwlA ΔcwlB This study
    HD(PcwlA-lacZ) HD73 strain containing plasmid pHTPcwlA This study
    HDΔsigK(PcwlA-lacZ) sigK mutant containing plasmid pHTPcwlA This study
    HDΔgerE (PcwlA-lacZ) gerE mutant containing plasmid pHTPcwlA This study
    HD(pHT-gfp-cwlB) HD73 strain containing plasmid pHT-gfp-cwlB This study
    HD(ΔcwlB::cwlB) HD(ΔcwlB) genetic complementation strain carrying pHTHFcwlB plasmid; Ermr This study
    HD(ΔcwlAΔcwlB::cwlB) HD(ΔcwlAΔcwlB) genetic complementation strain carrying pHTHFcwlB plasmid; Ermr Kanr This study
Plasmids
    pHT304-18Z Promoterless lacZ vector, Eryr Ampr, 9.7 kb 31
    pET-21b Expression vector, Ampr, 5.4 kb This laboratory
    pGEX-4T-1 Expression vector, Ampr, 4.9 kb 34
    pHT315 B. thuringiensis-E. coli shuttle vector 41
    pRN5101 Temp-sensitive plasmid, 8.0 kb 42
    pHTPcwlA pHT304-18Z carrying PcwlA, Ampr Ermr This study
    pETcwlB pET-21b containing cwlB gene, Ampr This study
    pGEXgerE Pgex-4T-1 containing gerE gene, Ampr This study
    pHT-gfp-cwlB pHT315 containing PcwlA-gfp-cwlB This study
    pRN5101ΩcwlA pRN5101 carrying partial cwlA deletion gene This study
    pRN5101ΩcwlB pRN5101 carrying partial cwlB deletion gene This study
    pRN5101ΩAB pRN5101 carrying partial cwlA cwlB deletion gene This study
    pHTHFcwlB pHT315 containing PcwlA-cwlB This study
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E. coli was cultured at 37°C in Luria-Bertani medium (1% NaCl, 1% tryptone, 0.5% yeast extract). B. thuringiensis strains were grown at 30°C in Schaeffer's sporulation medium (SSM) (27). The antibiotic concentrations used for bacterial selection were as follows: 100 μg/ml ampicillin for E. coli and 50 μg/ml erythromycin for B. thuringiensis.

DNA manipulation and transformation.

PCR was performed using Taq DNA polymerase and KOD DNA polymerase (New England BioLabs Ltd., Beijing, China). Amplified fragments were purified using Axygen, Inc., purification kits (Axgen Biotechnology Corporation, Hangzhou, China). Chromosomal DNA was extracted from E. coli and B. thuringiensis as described previously (28). Restriction enzymes and T4 DNA ligase (TaKaRa Biotechnology Corporation, Dalian, China) were used according to the manufacturer's instructions. Oligonucleotide primers (see Table S1 in the supplemental material) were synthesized by Sangon (Shanghai, China). Plasmid DNA was extracted from E. coli using the Axygen, Inc., plasmid extract kit (Axgen Biotechnology Corporation, Hangzhou, China). All constructs were confirmed by DNA sequencing (BGI, Beijing, China). Standard procedures were used for E. coli transformation. B. thuringiensis cells were transformed by electroporation as previously described (29).

Total RNA isolation and RT-PCR analysis.

Total RNA extraction from B. thuringiensis HD73 and reverse transcription-PCR (RT-PCR) identification were performed as described previously by Du et al. (30) unless otherwise noted. For RT-PCR analysis, the primers used to detect the expression of the cwlA and cwlB genes are shown in Table S1 in the supplemental material.

Determination of transcriptional start sites.

To determine the transcriptional start sites, we employed the SMARTer RACE (switching mechanism at the 5′ end of the RNA transcript-rapid amplification of cDNA ends) cDNA amplification kit (Clontech, Mountain View, CA) following the manufacturer's instructions. Gene-specific primers, CwlARace, and universal primer mix (UPM) were used to amplify the 5′ end of cwlB mRNA.

Construction of promoter PcwlA fusions with the lacZ gene.

The putative promoter fragment of PcwlA (102 bp), which is located upstream from the cwlA gene, was cloned from B. thuringiensis HD73 genomic DNA using the specific primers PcwlA-5 (with a PstI restriction site) and PcwlA-3 (with a BamHI restriction site). The PstI-BamHI fragment of the PcwlA promoter was then integrated into the vector pHT304-18Z, which harbors a promoterless lacZ gene (31). The recombinant plasmid pHTPcwlA was introduced into HD73 and the sigK mutant (8). The corresponding strains HD(PcwlA-lacZ) and HDΔsigK(PcwlA-lacZ) were selected by erythromycin resistance and PCR identification.

β-Galactosidase assays.

B. thuringiensis strains containing lacZ transcriptional fusions were cultured in SSM at 30°C and 220 rpm with appropriate antibiotics. Samples of 2.0 ml were taken from T8 to T20 at 1-h intervals (T0 is the end of the exponential phase; Tn is n hours after the end of the exponential phase). The cells were harvested, and the specific β-galactosidase activities of the samples were measured as previously described and expressed as Miller units per milligram of protein (32). The values reported are the means for at least three independent experiments.

Expression and purification of CwlB protein.

CwlB protein with a His tag was purified from E. coli. The expression plasmid pETcwlB was constructed by PCR amplification of the cwlB sequence from the B. thuringiensis HD73 genome using the primer pair cwlB-5 (with a BamHI restriction site) and cwlB-3 (with a SalI restriction site). The DNA fragment was digested with BamHI and SalI, cloned into pET21b (Novagen, Bloemfontein, South Africa) digested with the same restriction enzymes, and transferred into E. coli BL21(DE3) (33).

The E. coli BL21 strain harboring pETcwlB was grown to log phase in LB medium with ampicillin at 37°C. Expression of the CwlB-His protein was induced by adding IPTG (isopropyl-β-d-thiogalactopyranoside) to a final concentration of 2 mM, and the cultures were incubated for 3 h at 37°C. The cells were harvested by centrifugation at 13,500 × g for 10 min in 50-ml tubes and resuspended in 10 mM imidazole NPB buffer (10 mM imidazole, 1 M NaCl, 20 mM sodium phosphate, pH 7.4). Bacteria were lysed on ice by sonication using an ultrasonic cell disruption system. The bacterial lysate was centrifuged at 16,000 × g for 10 min at 4°C, and the supernatant contained the solubilized CwlB-His protein. The supernatant was filtered through a 0.45-mm-pore-size membrane filter (Nalgene) and loaded onto a HiTrap chelating column (1 ml; Pharmacia). After binding the protein, the column was washed with 10 mM imidazole NPB solution, and the target CwlB-His protein was eluted with NPB solution containing a stepwise gradient of imidazole from 100 to 500 mM.

Expression and purification of GerE.

GerE protein with a glutathione S-transferase (GST) tag was purified from E. coli. The expression plasmid pGEXgerE was constructed by PCR amplification of the gerE sequence from the B. thuringiensis HD73 genome using the primer pair gerE-5 (with a BamHI restriction site) and gerE-3 (with a SalI restriction site). The DNA fragment was digested with BamHI and SalI, cloned into pGEX-4T-1 (34) digested with the same restriction enzymes, and transferred into E. coli BL21(DE3).

When the optical density at 600 nm (OD600) reached 0.6, IPTG was added to a final concentration of 1 mM. After 4 h of induction at 37°C, bacterial cells were harvested by centrifuging the culture at 13,500 × g for 10 min. The pellet was resuspended with phosphate-buffered saline (PBS) buffer and sonicated on ice. All subsequent procedures were carried out at 4°C. The supernatant was collected by centrifuging the lysate at 13,500 × g for 20 min and loading it onto a glutathione-Sepharose 4B column previously equilibrated with PBS buffer. The column was washed with 50 mM Tris–HCl (containing 10 mM reduced glutathione, pH 8.0). The fractions were analyzed by SDS-PAGE. Fractions with the target protein were pooled and dialyzed against PBS buffer. The purified GST-GerE protein was analyzed by SDS-PAGE on a 12% polyacrylamide gel using a protein molecular standard. All the steps described above were performed according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Gel mobility shift assays.

GST-tagged GerE protein was purified from E. coli BL21(DE3) as described above. GerE-PcwlA binding experiments were performed using a modified gel mobility shift assay described previously (35). The DNA probe (1 mg) was incubated with various concentrations of purified GerE at 25°C for 20 min in a buffer containing 20 mM Tris base (pH 7.5), 2 mM dithiothreitol (DTT), 5 mM MgCl2, 0.5 mg ml−1 calf bovine serum albumin (BSA), and 5% (vol/vol) glycerol in a total volume of 20 μl. After incubation, nondenaturing 4% (wt/vol) polyacrylamide gels were stained with SYBR gold nucleic acid gel stain (Invitrogen) for 40 min in TBE (89 mM Tris-base, 89 mM boric acid, 1 mM EDTA, pH 8.0) buffer and photographed under UV transillumination using a Fuji X-5000.

Preparation of cell walls.

B. thuringiensis HD73 cells from exponential-phase cultures (absorbance at 600 nm of 1.5 to 2.0) growing in LB medium were deposited by centrifuging (16,000 × g for 5 min) at 4°C. All subsequent treatments were at 0 to 4°C. The bacteria were resuspended in a small volume of TK buffer (0.1 M Tris-HCl, 0.1 M KCl, pH 8.0) and disrupted with a BeadBeater (Biospec) in a vessel containing glass beads (0.1-mm diameter). After low-speed centrifugation (1,000 × g, 10 min) to remove the glass beads and unbroken cells, the crude cell walls were pelleted at 27,000 × g for 5 min at 4°C. The pellets were washed three times with TK buffer and resuspended in TK buffer (36).

Cell wall binding ability test.

The cell wall binding ability of the CwlB hydrolase enzyme was examined in distilled water containing the purified protein and B. thuringiensis cell walls. After 30 min of incubation at 0°C, the reaction mixture was centrifuged, and then the protein in the sediment was used for SDS-PAGE and Western blot analyses. Proteins in the samples were separated by electrophoresis (4% polyacrylamide stacking gel, 10% polyacrylamide separating gel) and then transferred onto polyvinylidene difluoride (PVDF) membranes before incubation in a 1:5,000 dilution of anti-His antibody (Proteintech Group, Inc., Chicago, IL, USA). Western blotting experiments were performed after the samples were separated by SDS-PAGE as described by Wang et al. (23).

Determination of the sites of cell wall cleavage by CwlB.

B. thuringiensis cell walls were suspended in 0.1 M TK buffer (adjusted to pH 7.0) to yield a final absorbance of 0.3 at 540 nm. Purified CwlB-His was added to the solution, followed by incubation at 37°C for 0, 10, 20, 40, or 60 min. One unit of cell wall hydrolase activity was defined as the amount of enzyme necessary to reduce the absorbance at 540 nm by 0.001 per min (37).

The amino groups released were labeled with 1-fluoro-2,4-dinitrobenzene during enzyme digestion of B. thuringiensis cell walls (38). The hydrolyzed dinitrophenyl (DNP) compounds were analyzed on a reverse-phase column. The release of free reducing groups during the enzymatic reaction was assayed as described by Thompson and Shockman (39).

Construction of GFP gene fusion directed by the CwlA promoter.

To determine the localization of the CwlB protein in B. thuringiensis, a fusion construct with the GFP gene was created by PCR amplification. A 669-bp DNA fragment of the cwlB ORF and 864 bp of the upstream sequence, including the 102-bp promoter, 714-bp GFP ORF, and 48-bp linker, were spliced in turn by overlapping PCR (see Fig. 7A). cwlB and promoter PcwlA were amplified by PCR using genomic DNA from B. thuringiensis HD73 as the template. The GFP ORF and linker fragment were amplified by PCR using the Cry1Ac-GFP plasmid as the template (40). The PstI-EcoRI fragment of GFP-cwlB was then ligated into the shuttle vector pHT315 (41). Recombinant pHT-gfp-cwlB was introduced into the B. thuringiensis HD73 strain by electroporation to obtain the corresponding strain HD(pHT-gfp-cwlB). The primers in this study are listed in Table S1 in the supplemental material.

Fig 7.

Fig 7

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Localization of the CwlB protein. (A) Construction strategy for the GFP gene fusion directed by the CwlA promoter. (B) Images of HD(pHT-gfp-cwlB) strain cells in SSM at T9, T12, T15, and T18. The white arrows, yellow arrows, and blue arrows indicate nonautolyzed cells, lysed cells, and crystal proteins, respectively. GFP, green fluorescent protein signal in the bacterial cells; FM4-64, red fluorescent signal of FM4-64 stain; overlay, green and red fluorescent signals; PC, phase-contrast microscopy. Bar, 7.5 μm. (C) GFP quantification analysis of cell walls from the HD(pHT-gfp-cwlB) strain and the wild-type HD73 strain. Error bars show standard deviations.

Laser confocal microscopy.

FM4-64, a vital membrane stain (Molecular Probes, Inc., Eugene, OR), was suspended in DMSO (dimethyl sulfoxide) at a final concentration of 100 μM liter−1. Bacterial cells were stained with FM4-64 (100 μM liter−1) for 1 min on ice. The stained cells were scanned using a confocal laser scanning microscope (Leica TCS SL; Leica Microsystems, Wetzlar, Germany).

GFP quantification in the B. thuringiensis cell walls.

The cell walls from the B. thuringiensis HD (pHT-gfp-cwlB) strain were collected for GFP quantification at T9, T12, T15, and T18, and the OD600 was determined by spectrophotometry. The GFP fluorescence of the B. thuringiensis cell walls was measured at an excitation wavelength of 476 nm and an emission wavelength of 507 nm using a fluorescence spectrophotometer (F-4500; Hitachi, Japan). For each specific measurement, 400 μl of each culture was placed into a cuvette and measured immediately. The relative fluorescence units of GFP were obtained by dividing the fluorescence value by the OD600 (40).

Construction of the cwlA, cwlB, and cwlAB deletion mutants.

All primers for gene deletion were designed according to the B. thuringiensis HD73 genome sequence. The 322-bp fragment upstream from the start codon of cwlA (cwlA fragment A) was amplified by PCR using B. thuringiensis HD73 genomic DNA as the template and CwlA-a and CwlA-b as primers. The primers CwlA-c and CwlA-d were used to amplify the 335-bp fragment downstream from the 107th codon of cwlA (cwlA fragment B), and the primers AKm-a and AKm-b were used to amplify a 1,473-bp kanamycin resistance gene (kan) cassette which is directed by the PaphA3 promoter from pDG780 (23). cwlA fragment A, Kan, and cwlA fragment B were ligated together by overlapping PCR using primers CwlA-a and CwlA-d. The resulting fragment (2,150 bp) was inserted into the BamHI-SalI restriction sites of the erythromycin-resistant, temperature-sensitive suicide plasmid pRN5101 (42) to generate the pRN5101ΩcwlA plasmid. The recombinant plasmid was electroporated into HD73. Transformants resistant to erythromycin and kanamycin were grown at 30°C and transferred to LB liquid medium with kanamycin at nonpermissive temperature (38°C) for about 10 generations. The bacterial cells were then plated onto LB agar plates supplemented with kanamycin or erythromycin, respectively. They were incubated for 12 h at 30°C. The transformants with kanamycin resistance but without erythromycin resistance were screened and identified by PCR; the final HD(ΔcwlA) strain, with a deletion from the start codon to the 107th codon of cwlA and insertion of the kanamycin resistance gene cassette at the same position, was verified by PCR; and DNA sequencing was performed (see Fig. S1 in the supplemental material).

The primers AB-a/AB-b/AB-c/AB-d and ABKm-a/ABKm-b were used to amplify the 382-bp fragment upstream from the start codon of cwlA (cwlA cwlB fragment A), the 1,473-bp kan gene cassette, and the 279-bp fragment downstream from the end codon of cwlB (cwlA cwlB fragment B). These fragments were ligated together by overlapping PCR using primers AB-a and AB-d. These were integrated into pRN5101 to generate pRN5101ΩAB. The corresponding deletion-insertion mutant, strain HD(ΔcwlAΔcwlB), with a deletion from the start codon of cwlA to the end codon of cwlB and the insertion of the kanamycin resistance gene cassette at the same position (see Fig. S1 in the supplemental material), was selected and confirmed by PCR, and DNA sequencing was performed.

The 788-bp fragment upstream from the 11th codon of cwlB (cwlB fragment A) was amplified by PCR using B. thuringiensis HD73 genomic DNA as the template and CwlB-a and CwlB-b as primers. The primers CwlB-c and CwlB-d were used to amplify the 882-bp fragment downstream from the end codon of cwlB (cwlB fragment B). Two fragments were ligated by overlapping PCR using the primers CwlB-a and CwlB-d. The PCR products were digested with BamHI-SalI restriction enzymes and then ligated with the plasmid pRN5101, which was digested with the same restriction enzymes, to generate the recombinant plasmid pRN5101ΩcwlB. This recombinant plasmid was transformed into the HD73 strain by electroporation. The Ermr transformants were verified by PCR using the CwlB-a and CwlB-d primers and cultured at the nonpermissive temperature of 38°C. Colonies with no erythromycin resistance were selected, and one deletion mutant, strain HD(ΔcwlB) (see Fig. S1 in the supplemental material), was verified by PCR and DNA sequencing.

Construction of the sigK and gerE disruption mutants.

The primer pairs SigK-a/SigK-b, SigK-c/SigK-d, and KKm-a/KKm-b were used to construct sigK deletion mutation cassettes, including the 606-bp fragment upstream from the start codon of sigK (sigK fragment A), the 1,473-bp kan gene, and the 551-bp fragment downstream from the end codon of sigK (sigK fragment B). These fragments were ligated together by overlapping PCR using primers SigK-a and SigK-d. The PCR products were digested with the BamHI and SalI restriction enzymes, and the digested fragments were purified and ligated with pRN5101 to generate pRN5101ΩSigK. The corresponding strain HD(ΔsigK), with a deletion from the start codon to the end codon of sigK and the insertion of the kanamycin resistance gene cassette at the same position, was selected and confirmed by PCR and DNA sequencing (8) (see Fig. S1 in the supplemental material).

The primers GerE-a/GerE-b and GerE-c/GerE-d were used to construct gerE deletion mutation cassettes, including the 1,064-bp fragment upstream from the start codon of gerE (gerE fragment A) and the 998-bp fragment downstream from the end codon of gerE (gerE fragment B). Two fragments were ligated by overlapping PCR using primers GerE-a and GerE-d. The PCR products were digested with the BamHI and HindIII restriction enzymes. The digested fragments were purified and ligated with pRN5101 to generate pRN5101ΩgerE. The corresponding HD(ΔgerE) deletion mutants (see Fig. S1 in the supplemental material) were selected and confirmed by PCR and DNA sequencing.

Genetic complementation of the cwlB deletion mutant.

The oligonucleotide primers cwlBF/cwlBR (with a SalI restriction site) and PcwlAF (with a HindIII restriction site)/PcwlAR were used to amplify the cwlB gene and its own promoter, PcwlA, respectively. The cwlB and PcwlA fragments were ligated by overlapping PCR using primers PcwlAF and cwlBR. The resulting fragment was digested with HindIII and SalI and then integrated into shuttle vector pHT315 (41) to generate pHTHFcwlB. The genetically complemented mutant strains HD(ΔcwlB::cwlB) and HD(ΔcwlAΔcwlB::cwlB) were obtained by introducing pHTHFcwlB into HD(ΔcwlB) and HD(ΔcwlAΔcwlB), respectively.

Quantification of Cry1Ac protein production.

The B. thuringiensis HD73 wild-type strain and the HD(ΔcwlB) mutant were grown in SSM medium at 30°C at 220 rpm. After complete autolysis, 1 ml of each sample was centrifuged, and the cells were suspended in 500 μl of Na2CO3 (50 mM, pH 8.0). The bacterial cells were then ruptured with a Mini-BeadBeater (Biospec Products, Inc., Bartlesville, OK, USA), and the supernatant was mixed with 3× protein-reducing sample buffer (32) and boiled for 5 min for subsequent total-protein quantitation and SDS-PAGE. The total protein quantitation was performed using the Pierce 660-nm protein assay reagent (Thermo Scientific). The same amounts of total protein were taken for SDS-PAGE of the HD73 and HD(ΔcwlB) mutants. Cry1Ac protein production was quantified with Image Master 1D Elite software (Amersham Biosciences).

Microscopic observation and determination of sporulation efficiency.

Cells were cultured in a conical flask containing 50 ml of SSM medium at 30°C. Samples of 1 ml were taken at T0, T16, T20, and T24 and centrifuged; the sediment was suspended with 100 μl deionized water. Amounts of 0.5 μl of the samples were placed on glass slides, and then cell morphology was observed by optical microscopy.

Sporulation efficiency was measured in SSM. The total amount of cells at T1 was determined. The spores released were collected at T24, and the cell suspension was heated to 65°C for 20 min to inactivate vegetative cells and then plated onto LB agar medium. The sporulation frequency was defined as the ratio of the number of colonies after heat treatment to the number of colonies at T1.

RESULTS

Sequence analysis and transcriptional units in the bt2492–bt2495 locus.

Two open reading frames, bt2493 and bt2494, in the B. thuringiensis HD73 genome were analyzed (Fig. 1A). The bt2494 protein is encoded by a 669-bp DNA fragment that was annotated as a putative cell wall autolysin. The protein's secondary structure (http://www.ncbi.nlm.nih.gov) indicates the existence of three predicted domains, two Src homology 3 (SH3) domains and a MurNAc-LAA family domain (Fig. 1B). This domain organization is very similar to that of Bacillus subtilis CwlC and CwlH (Fig. 1B), which have an N-terminal MurNAc-LAA domain and a C-terminal substrate-binding domain. bt2493, encoded by a 351-bp DNA fragment upstream from bt2494, contains only one domain of the MurNAc-LAA family and was also annotated as a putative cell wall autolysin. Thus, bt2493 and bt2494 were designated CwlA and CwlB. Orthologs of the cwlAB locus were found in many B. cereus and B. thuringiensis strains. The domains of CwlA and CwlB from B. thuringiensis HD73 were conserved in the genome sequences of B. cereus ATCC 14579, B. cereus B4264, B. cereus G9842, B. thuringiensis BMB171, B. thuringiensis 407, B. thuringiensis CT-43, B. thuringiensis HD-789, B. thuringiensis HD-771 (43–49), and Bacillus weihenstephanensis KBAB4 (50), and their cwlAB locus sequence similarities were above 87%. However, there was no occurrence of the cwlAB operon in Bacillus anthracis, Bacillus mycoides, or Bacillus pseudomycoides.

Fig 1.

Fig 1

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Analysis of the cwlAB operon in B. thuringiensis HD73. (A) Map of the bt2492–bt2495 locus in B. thuringiensis strain HD73. The positions of the fragments deleted from the chromosome to disrupt various genes are indicated. The bent arrow represents the promoter. Dashed lines with small black arrows annotated by letters correspond to RT-PCR amplicons (see lanes in panel C). Solid lines below ORFs indicate operons. (B) Domain organization of the B. thuringiensis HD73 CwlB and B. subtilis CwlB, CwlC, and CwlH cell wall hydrolases. Gray boxes represent cell wall binding domains. Black boxes define the MurNAc-LAA domains. (C) RT-PCR analysis of the bt2492–bt2495 locus in B. thuringiensis strain HD73. Negative controls consisted of RT-PCR with total RNA and 16S rRNA primers. Positive controls consisted of PCR with genomic DNA. Letters refer to the positions of the RT-PCR products in the locus as depicted in panel A. RNA was extracted from cells harvested at T12 in SSM.

There is no potential stem-loop present between cwlA and cwlB. To determine whether cwlB and cwlA with their flanking genes form one operon, seven pairs of primers were designed according to the bt2492–bt2495 gene sequences, including RT2492-5/RT2492-3, RTcwlA-5/RTcwlA-3, RTcwlB-5/RTcwlB-3, RT2495-5/RT2495-3, RTA92-5/RTA92-3, RTAB-5/RTAB-3, and RTB95-5/RTB95-3 (Fig. 1A; see also Table S1 in the supplemental material). RT-PCR was carried out with total RNA extracted at T12 from B. thuringiensis HD73 cultures grown in SSM (Fig. 1C). The results showed that mRNAs for all four genes were present (Fig. 1C). A mRNA overlapping the cwlA and cwlB genes was detected by RT-PCR (Fig. 1C). However, no mRNA overlapping cwlA and bt2492, as well as no mRNA overlapping cwlB and bt2495, was transcribed (Fig. 1C). This suggests that the cwlB and cwlA genes were transcribed together and form an operon, cwlAB.

Regulation of the cwlAB operon.

The transcriptional start site was confirmed to be a single 5′-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon according to the sequences of 12 random clones obtained by 5′-RACE-PCR (Fig. 2A).

Fig 2.

Fig 2

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Analysis of cwlAB operon promoter activity in strains HD73, HD(ΔgerE), and HD(ΔsigK). (A) Nucleotide sequence of the cwlAB operon in B. thuringiensis HD73. Annotations are shown in the 1,441-bp sequence, in which the elided sections are indicated by dots. The transcription initiation point is marked as +1. Codons with double underlines are the transcription start and transcription end codons for cwlA, and codons with single underlines are the transcription start and transcription end codons for cwlB. Longer underlined regions indicate the putative −35/−10 consensus sequence of the cwlAB operon promoter. (B) The levels of promoter-directed synthesis of β-galactosidase in the strains were determined at the indicated times after growing the cells in SSM at 30°C; Tn is n hours after the end of the exponential phase. Each value represents the mean for at least three independent replicates. Error bars show standard deviations.

To study the transcription and regulation of the promoter PcwlA in the cwlAB operon, a PcwlA-lacZ fusion was constructed and transformed into B. thuringiensis HD73, HD(ΔsigK), and HD(ΔgerE). A β-galactosidase assay indicated that PcwlA promoter activity in the HD73 strain increased sharply after T10, which suggested that it was expressed in the late stage of sporulation. The transcriptional activity of PcwlA was demonstrated to be abolished in the sigK mutant strain HD(ΔsigK) (Fig. 2B). Typical σK-dependent sequences (HDCA and CATANNNDD) (51) were also found to exist in the −35 and −10 regions upstream from the transcriptional start site of the cwlA operon (Fig. 2A). All these results indicate that the promoter PcwlA is controlled by the σK factor. Some σK-dependent genes were negatively or positively regulated by the GerE regulator in the late stage of sporulation. The activity of the PcwlA-lacZ fusion in the gerE mutant HD(ΔgerE) was tested. The results showed that β-galactosidase activity in HD(ΔgerE) was significantly decreased compared to that in HD73 (Fig. 2B). An electrophoresis mobility shift assay (EMSA) to detect GerE protein and PcwlA promoter complexes was performed; the results showed that no binding band was found on the gel. All these results reveal that the promoter PcwlA is indirectly regulated by the GerE protein.

Mutation of cwlB delays spore release.

cwlAB operon mutants, including cwlA mutant HD(ΔcwlA), cwlB mutant HD(ΔcwlB), and double mutant HD(ΔcwlAΔcwlB), were constructed (Fig. 1A). The growth rates of all mutants were similar to that of HD73. The morphologies of vegetative cells at T0 and sporulation cells at T16 were not significantly different in the mutants and HD73 (Fig. 3). Optical microscopy observations showed that HD(ΔcwlB) and HD(ΔcwlAΔcwlB) completely released their spores at T24, compared with T20 for HD73 and HD(ΔcwlA) (Fig. 3), which means that the mutation of cwlB delays spore release and cwlB plays a role in mother cell lysis.

Fig 3.

Fig 3

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Optical micrographs of B. thuringiensis HD73 (wild-type strain) cells and HD(ΔcwlA), HD(ΔcwlB), and HD(ΔcwlAΔcwlB) mutant cells at T0, T16, T20, and T24 of incubation in SSM medium at 30°C. Bar, 10 μm.

In order to verify that the morphology resulted from cwlB gene disruption, genetically complemented strains of the cwlB deletion mutant were constructed. The morphologies of vegetative cells of the HD(ΔcwlB::cwlB) and HD(ΔcwlAΔcwlB::cwlB) strains were not significantly different from that of HD73 at T0 (Fig. 4). However, although a few spores of the complemented strains were released at T16, no spores of the wild-type strain HD73 were released at that time, and almost all of the spores of the complemented strains were released at T20, the same as for HD73. The results confirmed that the delay of spore release resulted from the disruption of the cwlB gene.

Fig 4.

Fig 4

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Optical micrographs of the genetically complemented strains HD(ΔcwlB::cwlB) and HD(ΔcwlAΔcwlB::cwlB) at T0, T16, and T20 of incubation in SSM medium at 30°C. Bar, 10 μm.

The effects of cwlB mutation on sporulation and Cry protein production at T24 were analyzed in B. thuringiensis. The results showed that the sporulation rate of HD(ΔcwlB) was slightly decreased compared to that of HD73 (Fig. 5A). Cry protein production was determined in various strains by SDS-PAGE after crystal and spore release. The Cry protein production in HD(ΔcwlB) was similar to that in HD73 (Fig. 5B).

Fig 5.

Fig 5

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Comparisons of sporulation frequency and the production of crystal proteins between the HD(ΔcwlB) mutant and wild-type strain HD73. (A) Graph depicting sporulation frequency. Error bars show standard deviations. (B) Analysis of Cry1Ac crystal production in the wild-type strain HD73 and the mutant HD(ΔcwlB), as described in Materials and Methods.

Characterization of the CwlB protein.

CwlB contains a MurNAc-LAA family domain that acts on the amide bond between MurNAc and the N-terminal l-alanine residue of the stem peptide (Fig. 6A), and it is consequently annotated as an N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA). It was shown to play a role in mother cell lysis in B. thuringiensis, as described above. To confirm its cell wall binding ability and amidase activity in vitro, a CwlB-His tag fusion protein, which consisted of 233 amino acid residues, was expressed and purified. SDS-PAGE analysis showed that a single protein band with a molecular mass of 24.5 kDa, purified by nickel affinity chromatography, was found on the gel (Fig. 6B).

Fig 6.

Fig 6

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Characteristics of the CwlB protein. (A) Schematic diagram of the amide bonds cleaved by amidase. (B) SDS-PAGE analysis of CwlB protein (6 mg) expressed in E. coli (pETCwlB) and purified using nickel affinity column chromatography. M, protein marker. (C) Western blot analysis of cell wall binding ability. H, His tag. (D) Digestion of B. thuringiensis cell walls by purified CwlB protein. B. thuringiensis cell walls (2.5 mg) and purified CwlB (6 mg) were mixed in 5 ml TK buffer (pH 7.0) and then incubated at 37°C. Aliquots (500 μl of each) were removed at various intervals to determine turbidity at 540 nm (▲); the cell walls without protein were used for the negative control (♦); DNP-l-alanine (●) and DNP-d-alanine (■) were separately measured in reaction mixture with the cell walls and CwlB.

To examine the cell wall binding ability of CwlB, the purified CwlB-His protein and purified B. thuringiensis Cry1Ie-His protein (49, 50), which is an endotoxin with no ability to bind with cell walls and was used as the negative control, were added into B. thuringiensis cell wall buffer with a final absorbance of 0.3 at 540 nm. CwlB was also used in the absence of cell walls as another control to test whether CwlB can sediment. The reaction mixture was centrifuged at 0°C as described in Materials and Methods. Western blot analysis showed that a single protein band for CwlB was found in sediment using a His antibody, but no band was found for Cry1Ie-His or CwlB-His in sediment without cell walls (Fig. 6C). This means that the CwlB protein was bound to the B. thuringiensis cell walls.

As described above, the mixture containing CwlB protein and cell walls was incubated at 37°C, and the OD540 values were calculated. The results demonstrated that cell wall density decreased after CwlB-His was added (Fig. 6D). This suggested that CwlB-His digested the B. thuringiensis cell walls. To determine the peptidoglycan cleavage site, the free amino groups of the digested sample were investigated by labeling free amino groups with 1-fluoro-2,4-dinitrobenzene, followed by hydrolysis of glycoside and amido linkages of the cell walls with HCl. Finally, the dinitrophenyl (DNP)-amino acids were separated by reverse-phase high-performance liquid chromatography. After digesting for 60 min, only the amount of DNP-l-alanine increased during the enzyme reaction (Fig. 6D). All of these results indicate that CwlB is an N-acetylmuramoyl-l-alanine amidase.

Localization of the CwlB protein.

The CwlB protein, which is composed of two C-terminal SH3 domains, was shown to act on the cell wall peptidoglycan of B. thuringiensis. It was necessary to determine the localization of the CwlB protein in B. thuringiensis cells to elucidate the role of the CwlB protein in mother cell lysis. A fusion of the GFP gene with the cwlB gene at the 5′ terminus, GFP-cwlB, was constructed in the vector pHT315 to obtain pHT-gfp-cwlB and then transformed into B. thuringiensis HD73 (Fig. 7A). The resulting strain, HD(pHT-gfp-cwlB), was cultured in SSM at 30°C and 220 rpm with appropriate antibiotics. The vital cell membrane of B. thuringiensis was stained with FM4-64. Cells were observed via laser confocal microscopy. A red fluorescent signal indicated the membrane of a bacterial cell, and a green fluorescent signal indicated the location of the Gfp-CwlB fusion protein.

At stage T9, bipyramidal crystals and spores were observed but no green fluorescent signal was detected in B. thuringiensis cells. At stage T12, the cell walls were still intact and weak green fluorescent signals were observed in most of the mother cells of HD(pHT-gfp-cwlB). At stage T15, some cells had undergone autolysis and the green fluorescent signal intensities had increased significantly and were spread over the cell envelopes of nonautolyzed cells only (Fig. 7B, white arrows). Moreover, no green fluorescence was detected in lysed cells (Fig. 7B, yellow arrows). However, at stage T18, many crystals and spores were released and, although green fluorescence intensity was not diminished, no green fluorescence was detected in the crystals and spores (Fig. 7B, blue arrows). Additionally, we obtained the cell walls of B. thuringiensis HD73(pHT-gfp-cwlB) in SSM at T9, T12, T15, and T18 and calculated the relative fluorescence units (Fig. 7C). Fluorescence intensity was not evaluated at T9, but it was tremendously increased at T15 compared with that in the HD73 strain. These results suggest that the CwlB protein is localized on the cell envelope.

DISCUSSION

We identified a novel N-acetylmuramoyl-l-alanine amidase gene involved in B. thuringiensis mother cell lysis, which was named cwlB in this study. In B. subtilis, the results of prior studies of the involvement of hydrolase genes in mother cell lysis have been clear and definite. Three autolysins, CwlB, CwlC, and CwlH, were present at the time of mother cell lysis (16–19). We tried to search for the orthologs of CwlB, CwlC, and CwlH in B. thuringiensis. No amidase was found to share more than 41.7% sequence similarity with them or have the same domain organization as CwlB, CwlC and CwlH in B. thuringiensis. Sequence analysis showed that CwlB of B. thuringiensis in this study shares low sequence similarity (below 10.0%) with CwlB, CwlC, and CwlH of B. subtilis. However, they all consist of a MurNAc-LAA family domain and peptidoglycan binding domains which are different from each other (Fig. 1B). This suggests that CwlB of B. thuringiensis is a novel hydrolase involved in mother cell lysis. The cwlC and cwlH genes were σK dependent and expressed only in the late stage of sporulation in B. subtilis. The cwlB from B. thuringiensis showed a similar transcription pattern. Although single inactivation of cwlB, cwlC, and cwlH in B. subtilis has no effect on mother cell lysis (52), CwlB in B. thuringiensis was shown to play a role in mother cell lysis and delay spore release, even though CwlB shows low sequence similarity to the three autolysins in B. subtilis. This is the first report in which an autolysin gene in the B. cereus group is shown to be involved in mother cell lysis.

Although the disruption of cwlB was demonstrated to delay spore and crystal release in B. thuringiensis, mother cells still lysed after T24. This suggests that other hydrolases exist and participate in this cellular process. The combined effects of hydrolases have been shown to be required for many cellular functions. In E. coli, the endopeptidases penicillin-binding protein 4 (PBP 4), PBP 7, and MepA contribute to cleavage of the septum. However, when deletions in amidases are combined with deletions in lytic transglycosylases or endopeptidases, bacterial chains become very long and may contain up to 100 cells during cell division (53, 54). In B. subtilis, cell separation is greatly affected by a combination of two endopeptidases (38, 55, 56). Vegetative cell lysis is affected by the major autolysin, CwlB (57, 58), and greatly by four autolysins, including minor ones (CwlF [LytE] and CwlE [LytF]), in B. subtilis (38). Motility and cell wall turnover were also found to be affected by combinations of cell wall hydrolases in B. subtilis (58–60), and germination was found to be completely blocked in the absence of two deduced class II amidases (61). The combined effect of hydrolase deletion on mother cell lysis has been observed, and lysis was blocked in a mutant with the cwlB, cwlC, and cwlH genes inactivated (52). Therefore, to elucidate the mechanism of mother cell lysis in B. thuringiensis, we need to search for more cell wall hydrolases that combine with CwlB to act at the late stage of sporulation.

In this study, we reported that a novel cell wall hydrolase delays spore release and has no significant effect on sporulation and Cry protein production. It is a good candidate gene for disruption to develop a novel B. thuringiensis agent with greater persistence. This finding helps us to better understand the mechanism of mother cell lysis in B. thuringiensis and provides a foundation for developing a novel B. thuringiensis agent with protection of the insecticidal crystal proteins from UV degradation to increase their persistence.

Supplementary Material

Supplemental material
supp_195_12_2887__index.html (1.1KB, html)

ACKNOWLEDGMENTS

This work was supported by grants from the Key Project of Chinese National Programs for Fundamental Research and Development (973 program) (grant no. 2009CB118902), the National Natural Science Foundation (grant no. 31070083), and the National High Technology Research and Development Program of China (863 program) (grant no. 2011AA10A203).

Footnotes

Published ahead of print 19 April 2013

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00112-13.

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