Bacillus thuringiensis peptidoglycan hydrolase SleB171 involved in daughter cell separation during cell division

Abstract

Whole-genome analyses have revealed a putative cell wall hydrolase gene (sleB171) that constitutes an operon with two other genes (ypeB and yhcN) of unknown function in Bacillus thuringiensis BMB171. The putative SleB171 protein consists of 259 amino acids and has a molecular weight of 28.3 kDa. Gene disruption of sleB171 in the BMB171 genome causes the formation of long cell chains during the vegetative growth phase and delays spore formation and spore release, although it has no significant effect on cell growth and the ultimate release of the spores. The inseparable vegetative cells were nearly restored through the complementation of sleB171 expression. Real-time quantitative polymerase chain reaction analysis revealed that sleB171 is mainly active in the vegetative growth phase, with a maximum activity at the early stationary growth phase. Western blot analysis also confirmed that sleB171 is preferentially expressed during the vegetative growth phase. These results demonstrated that SleB171 plays an essential role in the daughter cell separation during cell division.

Introduction

Gram-positive Bacillus are a group of spore-forming bacteria with rigid cell walls composed primarily of the polymer peptidoglycan (PG) that maintains cell wall integrity and rigidity as well as protects the cells from various environmental stresses [1]. Peptidoglycan hydrolases (PGHs), some of which are referred to as autolysins and trigger cell lysis, a variety of hydrolases that cleave covalent bonds in the PGs of vegetative cells or spores [2], are found to involve in many cellular physiological processes, such as cell division, cell wall turnover, biofilm formation, cell-to-cell surface adhesion, genetic competence, protein secretion, and pathogenicity [3,4]. Moreover, PGHs have also been implicated in other biological activities, such as scaffolding surface proteins, adsorbing pathogens, and modulating host immune systems [2,5].

Bacillus thuringiensis has been recognized as an important entomopathogen because of its ability to produce insecticidal crystal proteins (ICPs), which are larvicidal proteins that are lethal to a wide range of pests [6]. During the late phase of sporulation, ICPs assemble into various conformational crystals, and these crystals and spores that are transformed from vegetative cells are released under the action of certain PGHs. This unique property has led to the successful use of B. thuringiensis in agriculture since the last century. However, the exposed ICPs may become inactivated under adverse conditions in the field, such as sunlight ultraviolet (UV) irradiation [7,8], which is a technical problem that affects the stability and persistence of B. thuringiensis-derived biopesticides. Thus, PGHs could be key parameters that manage the release of ICPs from mother cells. Therefore, investigation of the PGHs involved in cell lysis and cell division is of critical importance for improving the persistence and efficacy of B. thuringiensis biopesticides in agricultural environments.

At least 35 PGHs have been identified in B. subtilis, and these enzymes can be categorized into seven diverse groups based on their PG cleavage site [4,9]. Most of the previously characterized B. subtilis PGHs have a modular structure consisting of a cell wall binding domain and a catalytic domain which contain the enzyme active site [2,10,11]. These PGHs are involved in cell morphogenesis and cell wall turnover and have been extensively investigated [4,12,13]. Various PGHs have also been identified in other Bacillus members, including B. anthracis, B.cereus, and B. megaterium [2,13,14]. Among these PGHs, SleB is known as a spore cortex-lytic enzyme (SCLE), which has been implicated in full cortex hydrolysis and spore germination of B. subtilis [15–17], B. anthracis [18–21], B. cereus [22], and B. thuringiensis [23,24]. SleB has been identified as a lytic transglycosylase that hydrolyzes the bond between N-acetylmuramic acid and N-acetylglucosamine [18].

In the present study, we focused on a Bacillus vegetative cell-active SleB gene, sleB171, which consists of an operon with two other genes in the B. thuringiensis BMB171 genome. The sleB171 gene was cloned, expressed, and purified to obtain the SleB171 protein. Through gene disruption and expression complementation of sleB171, we demonstrated that SleB171 was involved in the daughter cell separation during cell division of B. thuringiensis BMB171 cells. Our results also confirmed that sleB171 is preferentially expressed during the vegetative growth phase.

Materials and Methods

Bacterial strains and culture conditions

Escherichia coli strains DH5α [supE44ΔlacU169(Φ80 lacZΔM15) hdsR17 recA1 endA1 gyrA96 thi-1 relA1] (TaKaRa, Dalian, China) and BL21(DE3) [F⁻ dcm ompT hsdS(r_B⁻ m_B⁻) gal λ(DE3)] were used to construct the recombinant plasmids and express the SleB171 protein, respectively. Bacillus thuringiensis strain BMB171 [25] was used as the host strain for polymerase chain reaction (PCR) amplification, gene disruption, and expression complementation of the PGH-encoding gene sleB171. The E. coli strain BL21(DE3) harboring recombinant plasmid pMB406 was named E. coli MB406. The sleB171-disrupted B. thuringiensis BMB171 mutant strain was named BMB171ΔsleB171, and the derivative strain harboring recombinant plasmid pMB404 was named B. thuringiensis MB404.

The B. thuringiensis and E. coli strains were grown in lysogeny broth (LB) medium [26] at 30 and 37°C, respectively. In addition, B. thuringiensis MB404 was grown in LB supplemented with erythromycin (Erm) at a final concentration of 25 μg/ml. Escherichia coli MB406 and the B. thuringiensis BMB171ΔsleB171 mutant strain were grown in LB supplemented with kanamycin (Kan) at final concentrations of 50 and 25 μg/ml, respectively.

Cloning, expression, and purification of recombinant SleB171 protein

The plasmids and oligonucleotide primers used in this study were listed in Table 1. Genomic DNA was isolated from B. thuringiensis BMB171 using the method of Gonzalez et al. [29]. The recombinant plasmids harboring the full-length or truncated sleB171 gene were schematically illustrated in Supplementary Fig. S1. Briefly, the full-length sleB171 gene was amplified from the B. thuringiensis BMB171 genome using the primers SS1 and SS2. The 798-bp PCR-amplified fragment was sequenced prior to digestion with XbaI and PstI. The digested fragment was subsequently ligated to the XbaI/PstI double-digested site of the previously constructed plasmid pMB164 [28], which yielded the recombinant plasmid pMB404. To construct the recombinant plasmid pMB406 harboring the truncated sleB171 gene lacking a 99-bp putative signal peptide-encoding sequence, the sleB171 fragment was amplified from pMB404 using the primers S-1 and S-2. The resulting fragment was digested with EcoRI and HindIII and subsequently ligated to the EcoRI/HindIII site of the E. coli expression vector pET-28a(+) (Novagen, Gibbstown, USA) to generate the recombinant plasmid pMB406 (Supplementary Fig. S1).

Table 1.

Plasmids and oligonucleotide primers used in this study

Plasmids or primers	Phenotypes or sequencesa	Sources or references
Plasmids
pDG780	AmprKanr, plasmid vector carrying Kanr expression cassette; 4445 bp	[27]
pMB146	AmprErmr, carrying B. thuringiensis constitutive promoter Pcry3Aa; 8466 bp	[28]
pET-28a(+)	Kanr, E. coli expression vector, 5369 bp	Novagen
pMB404	AmprErmr, pMB146 derivative harboring Pcry3Aa and full-length sleB171, 8113 bp	This study
pMB406	Kanr, pET-28a(+) derivative harboring the truncated sleB171, 6066 bp	This study
Oligonucleotide primersb
SS1	5′-CTGTCTAGAATGCGCCAAAAAGCTA-3′ (XbaI)
SS2	5′-AAACTGCAGCTATTTACAGAAAATATG-3′ (PstI)
S-1	5′-ACGGGAATTCATGATACAACTAAAGAATGTA-3′ (EcoRI)
S-2	5′-CCCAAGCTTCTATTTACAGAAAATATG-3′ (HindIII)
SDM1	5′-ATGCGCCAAAAAGCTATGTTTAA-3′
SDM2	5′-TATTCTCATTTTAGCCATCTGCGTTTGCCATCAG-3′
SDM3	5′-ATGGCTAAAATGAGAATATCAC-3′
SDM4	5′-CTAAAACAATTCATCCAG-3′
SDM5	5′-CTGGATGAATTGTTTTAGTATACGGAGAATCACGCGG-3′
SDM6	5′-GCACGTTGATAGCTATTTTCCGCT-3′
16SF	5′-AGCGAATGGATTAAGAGCTT-3′
16SR	5′-AAATGTTATCCGGTATTAGC-3′

^aAmp^r, ampicillin resistance; Erm^r, erythromycin resistance; Kan^r, kanamycin resistance; cry3Aa, a B. thuringiensis insecticidal gene; P_cry3Aa, the promoter of cry3Aa; sleB171, a B. thuringiensis BMB171 peptidoglycan hydrolase gene.

^bThe underlined sequences indicate the restriction enzyme sites.

The transformation of recombinant plasmid pMB406 into E. coli BL21(DE3) was performed using a standard method [26]. Recombinant E. coli MB406 cells harboring pMB406 were grown in LB medium containing 50 μg/ml of Kan at 37°C until the optical density (OD) at 600 nm (OD₆₀₀) reached 0.6. Then isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.2 mM to the culture to induce the expression of recombinant SleB171. After further incubation for 7 h at 30°C, the cells were harvested through centrifugation and subsequently treated through sonication. The suspension was centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was collected. Recombinant SleB171 was purified from the supernatant using a nickel-nitrilotriacetic acid spin column (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Construction of the BMB171ΔsleB171 mutant

To construct recombinant gene fragment nsleB171-kan^r-csleB171 for the homologous double exchange of sleB171 gene fragments in the B. thuringiensis genome (Fig. 1B), four separate PCR reactions were performed using the standard Splicing by Overlap Extension (SOE) method [26]. Briefly, 455-bp N-terminal sequence of sleB171 (nsleB171) and 478-bp C-terminal sequence of sleB171 along with partial sequence of ypeB (csleB171/ypeB′) fragments were amplified from the B. thuringiensis BMB171 genome using the primers SDM1/SDM2 and SDM5/SDM6, respectively; a 795-bp encoding sequence of Kan resistance encoding cassette (Kan^r) was amplified from the plasmid pDG780 [27] using the primers SDM3 and SDM4. A fused 1232-bp nsleB171-Kan^r fragment was subsequently amplified using the primers SDM1 and SDM4 and the previously amplified overlapping fragments nsleB171 and Kan^r as heteroduplex templates. Moreover, the 1692-bp full-length ‘nsleB171-kan^r-csleB171’ was amplified using the primers SDM1 and SDM6 and the previously amplified overlapping fragments nsleB171-Kan^r and csleB171/ypeB′ as heteroduplex templates, which included the 436-bp nsleB171, 795-bp Kan^r, and 461-bp csleB171/ypeB¢ fragments (Fig. 1Ba). The 1692-bp linear fragment was transferred into B. thuringiensis cells through electroporation using a previously described method [30], resulting in a sleB171-disrupted mutant through a homologous double-crossover strategy (Fig. 1Ba, b), BMB171ΔsleB171, with the Kan^r cassette inserted into the sleB171 encoding frame, but without altering the downstream ypeB and yhcN (Fig. 1Bc) genes. For screening and identification of the BMB171ΔsleB171 mutant, the colonies resistant to Kan were selected from the plates. Their genomic DNAs were prepared separately and then used as template DNAs for the PCR amplifications of the fused gene fragments ‘nsleB171-Kan^r’ and ‘Kan^r-csleB171/ypeB′’ using the primer pairs SS1/SDM4 and SDM3/SDM6, respectively, and Kan^r or sleB171 gene alone using primer pairs SDM3/SDM4 and SS1/SS2, respectively.

Figure 1.

Schematic presentation of the structural organization of the sleB171 operon and flow chart of the B. thuringiensis BMB171DsleB171 mutant construction In (A): (a) sleB171 operon and (b) SleB171 protein. In (B): (a) constructed fusion gene fragment using the SOE strategy, (b) sleB171 operon in B. thuringiensis BMB171 genome, and (c) the resulting sleB171 operon mutant in the B. thuringiensis BMB171ΔsleB171 genome. AA, amino acid; SS, signal sequence; P, promoter; PG, peptidoglycan.

Analytical assays

To measure the growth curves of B. thuringiensis BMB171 and BMB171ΔsleB171, the cells were inoculated onto LB plates and incubated for 10 days. The cultures were harvested, resuspended in sterile phosphate-buffered saline (PBS; pH 7.0), and stored at −20°C for 48 h to facilitate complete spore formation. After the OD₆₀₀ was adjusted to 0.8 with sterile PBS buffer (pH 7.0), a 100-μl aliquot of spore suspension was inoculated and cultured in 500-ml Erlenmeyer flasks containing 100 ml of LB medium at 210 rpm and 37°C. The cell density was measured at 600 nm using an ultraviolet/visible (UV/VIS) spectrophotometer (DU-800 Nucleic Acids/Protein Analyzer; Beckman Coulter, Danvers, USA). The purified SleB171 protein was quantified according to the Bradford method [31]. The expression of SleB171 in E. coli MB406 was analyzed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels according to the Laemmli method [32].

Microscopy

Bacillus thuringiensis cells were grown overnight on an LB plate at 30°C, and the cells were then harvested and resuspended in sterile PBS (pH 7.0) to an OD₆₀₀ of 0.8. Each 50-μl aliquot of cell suspension was inoculated into 100-ml fresh LB medium in a 500-ml Erlenmeyer flask. The cells were subject to continuous incubation for 144 h at 200 rpm and 30°C. At 24-h intervals, a 20-μl cell sample was obtained. The Gram-stained cells were subsequently examined using a 100 × oil immersion objective lens on a phase-contrast microscope (Olympus BX51, Tokyo, Japan). The cell samples collected within 48 h were examined using a JSM-6390/LV scanning electron microscope (SEM; NTC, Tokyo, Japan) and H-7650 transmission electron microscope (HITACHI, Tokyo, Japan) according to the manufacturer's instructions.

Real-time quantitative PCR

At each 24-h interval, total RNA was extracted from B. thuringiensis BMB171 and the BMB171ΔsleB171 cell samples under the above incubation conditions using a TRIzol kit (Invitrogen, Carlsbad, USA). The cDNA synthesis was performed using the Revert Aid™ First-strand cDNA synthesis kit (Fermentas, Waltham, USA) according to the manufacturer's instructions. Real-time quantitative PCR was performed as previously described [33] to measure the mRNA levels. The reactions were performed in triplicate. As an internal control, the 16S rRNA gene was amplified using the primers 16SF and 16SR (Table 1). The comparative cycle threshold method ($2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\mathrm{T}}}$ method) was used to analyze the mRNA levels [34].

Sleb171 antiserum preparation

Purified SleB171 protein was subject to 10% SDS-PAGE. The gel was stained minimally with Coomassie blue; the sole band corresponding to SleB was excised and the protein was electroeluted from gel. The electroeluted protein solution was dialyzed exhaustively against distilled water and lyophilized. Approximate 1 mg purified SleB171 protein was subcutaneously injected into the neck region of a New Zealand rabbit after being emulsified with Freund's complete adjuvant. Three subsequent booster injections were given at Days 14, 21, and 28. The antiserum was collected 10 days after the last injection.

Western blot analysis

Bacillus thuringiensis BMB171 and BMB171ΔsleB cells under the above incubation conditions were collected at each 24-h interval. Cells were washed twice with PBS (pH 7.0) and were adjusted to an OD₆₀₀ of 0.8. Each 500 μl cell suspension was treated by sonication. The whole cell fractions were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Western blotting was performed as previously described [35], except that polyclonal SleB171 antiserum was used as the primary antibody.

Database search

The sleB171 sequence was characterized by conducting BLASTN and BLASTP searches on the GenBank nucleotide and amino acid (aa) sequence database using the National Center for Biotechnology Information (NCBI) server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The conserved domain architectures of SleB171 were analyzed using the NCBI online tool ‘Conserved Domain Search’ (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Results

Cloning, expression, and purification of SleB171

A whole-genome analysis of the B. thuringiensis BMB171 genome (GenBank accession number: CP001903.1) revealed the presence of an operon consisting of three genes, sleB, ypeB, and yhcN, which were originally annotated to encode a putative SCLE, a putative membrane-binding protein, and an unknown protein, respectively [36]. The amino sequence alignment analysis (Supplementary Fig. S2) showed that the ‘SleB’ protein of B. thuringiensis BMB171 shares over 99% sequence identity with the SleB protein of B. cereus ATCC10876 (GenBank accession number: ZP04317839.1), the CwlJ protein of B. cereus ATCC 14579 (GenBank accession number: NP832505.1) [37], the SceA protein of B. thuringiensis YBT-1520 (EMBL Nucleotide Sequence Database accession number: FR728432) [38], and the SleB protein of B. thuringiensis CGMCC 1.1752 (GenBank accession number: DQ523224.1). To distinguish the ‘sleB’ of B. thuringiensis BMB171 from other Bacillus sleB genes, this ‘sleB’ gene was renamed ‘sleB171’, and the operon was named ‘sleB171 operon’ (Fig. 1Aa) in the present study. The predicted full-length SleB171 protein is composed of 259 aa residues with a calculated molecular mass of ∼28.3 kDa and a pI of 9.67. These residues are composed of two distinctive domains (Fig. 1Ab): an N-terminal domain (from aa 43 to 99) with PG-binding activity and a C-terminal domain (from aa 159–259) with PG-hydrolysis activity. In the N terminus, the first 33 residues were predicted to be a signal sequence (SS) using SignalP (http://www.cbs.dtu.dk/services/SignalP/), and a possible cleavage site locates between aa 33 and 34.

For the expression of sleB171 in either B. thuringiensis or E. coli, the full-length sleB171 (780 bp) and truncated variant (733 bp) devoid of the SS were amplified and constructed into the recombinant plasmids pMB404 (Supplementary Fig. S1A) and pMB406 (Supplementary Fig. S1B), where the expression of full-length or truncated sleB171 was derived from either the constitutive promoter P_cry3Aa (pMB404) or the IPIG-inducible promoter P_t7 (pMB406), respectively. The SDS-PAGE analysis demonstrated that SleB171 was expressed in recombinant E. coli MB406 cells harboring pMB406 with the predicted size (∼28.3 kDa) (Supplementary Fig. S3, Lane 2, indicated by arrow); however, this protein was not observed in the BL21(DE3) profile (negative control) (Supplementary Fig. S3, Lane 1). The expressed SleB171 in the recombinant E. coli BL21(DE3) was purified and detected by SDS-PAGE (Supplementary Fig. S3, Lane 3, indicated by arrow). The band corresponding to SleB171 was eluted and used for the preparation of polyclonal antiserum.

Identification of the BMB171ΔsleB171 mutant

For the identification of the constructed BMB171ΔsleB171 mutant, the chimeric gene ‘nsleB171-Kan^r-csleB171’ was cross-checked through PCR using different primer combinations. As shown in Supplementary Fig. S4, the ‘nsleB171-Kan^r’ (1231 bp) and ‘Kan^r-csleB171/ypeB′’ (1256 bp) fragments occurred in the BMB171ΔsleB171 genome (Lanes 1 and 3, respectively) but not in the BMB171 genome (Lanes 2 and 4, respectively). The ‘Kan^r’ gene (795 bp) occurred in the BMB171ΔsleB171 genome (Lane 5) but not in the BMB171 genome (Lane 6); the ‘sleB171’ amplification fragments occurred as expected at 1561 bp in the BMB171ΔsleB171 genome (Lane 7) and at 730 bp in the BMB171 genome (Lane 8). All of the amplified bands were consistent with the predicted results, verifying that the sleB171-disrupted mutant BMB171ΔsleB171 was successfully constructed.

Microscopic examination of the BMB171ΔsleB171 phenotypes

The phenotypes of the BMB171ΔsleB171 mutant and wild-type BMB171 strains were examined and compared. The growth curve of BMB171ΔsleB171 was not affected by the sleB171 disruption compared with that of the wild-type strain (Supplementary Fig. S5), verifying that the mutation eliminating SleB171 did not affect the overall cell growth initiated from spores. However, the BMB171ΔsleB171 daughter cells remained inseparable during cell division and formed long cell chains during the vegetative growth phase, and the release of spores was significantly delayed. As shown in Fig. 2A, single cells could not be distinguished in the BMB171ΔsleB171 culture at 24−48 h. Most of the spores from the mother cells of BMB171ΔsleB171 were released at 120 h, which is inconsistent with that of the wild-type BMB171 cells that formed separate single cells over the 24−48 h growth phase with most of the spores released from the mother cells at 72 h (Fig. 2B). An SEM was used to further visualize the individual BMB171 cells (Fig. 3Aa) and long cell chains of the BMB171ΔsleB171 mutant (Fig. 3Ab). Transmission electron microscopy (TEM) clearly showed the septa formed in either vegetative cell types (Fig. 3Bc) or cell-embedded spores (Fig. 3Bd) of the BMB171ΔsleB171 mutant. However, the inseparable cells of the BMB171ΔsleB171 mutant (Fig. 4A) were almost restored to a wild-type BMB171 phenotype through sleB171 expression complementation in the recombinant BMB404 cells (Fig. 4B). These results indicated the involvement of SleB171 in cell separation and longer chain formation in the BMB171ΔsleB171 mutant, reflecting the inactivation of the sleB171 gene.

Figure 2.

Phenotypes of B. thuringiensis BMB171ΔsleB171 and BMB171 vegetative cells and spores at various growth phases under phase-contrast microscopy (A) BMB171ΔsleB171 and (B) BMB171.

Figure 3.

SEM and TEM micrographs of B. thuringiensis BMB171 and BMB171ΔsleB171 vegetative cells or spores In (A): (a) BMB171 vegetative cells under 48 h incubation and (b) BMB171ΔsleB171 vegetative cells under 48 h incubation. In (B): BMB171 vegetative cells (a) and spores (c) under 48 and 72 h of incubation, respectively, and BMB171ΔsleB171 vegetative cells (b) and embedded spores (d) under 48 and 72 h of incubation, respectively. S, spores.

Figure 4.

Phase-contrast microscopic micrographs of B. thuringiensis BMB171ΔsleB171 and MB404 cells under 48  h incubation (A) BMB171ΔsleB171 cells and (B) MB404 cells (BMB171ΔsleB171 expressing SleB171).

Expression of sleB171 operon genes at different growth stages

The genes sleB171, ypeB, and yhcN in the sleB171 operon were subject to a differential expression activity analysis using quantitative real-time PCR. As expected, no sleB171 activity was detected in the BMB171ΔsleB171 mutant across the entire 144-h growth phase, whereas in the wild-type BMB171 strain, the activity of sleB171 was relatively low in the first 48 h (the vegetative growth phase), rapidly increased to a maximum value at 72 h (the early stationary growth phase) and then decreased to low activity at 96–144 h (Fig. 5). Meanwhile, ypeB exhibited relatively substantial activity from 48 to 144 h of the growth phase in wild-type BMB171, with maximum activity at 96 h (the stationary growth phase); the yhcN gene exhibited temporary high activities from 72 to 96 h of the growth phase, with maximum activity at 72 h. To characterize the intracellular amount of SleB171 in wild-type BMB171 and BMB171ΔsleB171 cells at different growth phases, western blot analysis of SleB171 in the total cellular proteins extracted from both BMB171 and BMB171ΔsleB171 vegetative or sporulating cells was performed. SleB171 was expressed and retained at different levels in various growth phases of BMB171, with the highest level at 72 h (Fig. 6A, Lanes 1–6), consistent with the results of the gene activity assays (Fig. 5), whereas no band occurred on BMB171ΔsleB171 profile (Fig. 6B, Lanes 1–6). These results indicated that the SleB171 was preferentially vegetative cell active and primarily functioned in the physiological processes corresponding to daughter cell separation of cell division in the wild-type BMB171 strain.

Figure 5.

Real-time quantitative PCR analysis of the transcriptional activities of sleB171 operon genes at different cell growth phases

Figure 6.

Western blot analysis of SleB171 protein in B. thuringiensis BMB171 (A) and BMB171ΔsleB171 (B) intact cells under different growth phases Lanes 1–6, samples taken at 24, 48, 72, 96, 120, and 144 h, respectively; Lane 7, the purified SleB171 (as the positive control).

Discussion

Many previous investigations have revealed that the sleB from certain Bacillus strains constitutes a bicistronic operon with the ypeB gene, which cooperatively contributes to complete spore cortex hydrolysis during spore germination [15,39]. In the present study, the sleB171 was found to constitute an operon with two other genes (ypeB and yhcN) in the B. thuringiensis BMB171 strain. To determine whether other B. thuringiensis subspecies/strains also harbored the sleB171-like operon, PCR amplification was performed on the sleB171-like genes and sleB171 operon in 18 wild-type strains from 4 different B. thuringiensis subspecies, which identified that 13 strains had amplification bands corresponding to the sleB171 gene and 9 strains had amplification bands corresponding to the operon (data not shown), verifying the relatively conservative distribution of sleB171 along with the sleB171 operons in various B. thuringiensis wild-type strains.

The SleB and CwlJ have been identified as cortex hydrolases crucial for spore germination in different Bacillus species [15,18,20,24]. However, in B. subtilis and other Bacillus species, the CwlJ protein is expressed under the control of the mother cell sporulation factor σ^E and SleB is produced within the developing spore and controlled through σ^G in the forespore [20]. Although the sleB171 gene of B. thuringiensis BMB171 shares high sequence similarities with other Bacillus sleB and cwlJ genes, respectively (Supplementary Fig. S2), the results of the present study suggest that SleB171 is a significantly vegetative cell-active PGH (Figs 3, 5, and 6), which is inconsistent with the characteristics of known SleBs in Bacillus species. Therefore, we recognized this protein as a vegetative cell-active PGH. Moreover, the genes ypeB and yhcN in the sleB171 operon were previously annotated as a putative membrane-binding protein and an unknown protein, respectively [36]. However, the identities and biological functions of these proteins remain uncertain. In the present study, we demonstrated that the expressions of these proteins were remarkably increased during the late exponential phase and the spore-release phase. Therefore, these genes may play a physiological role in cell division, particularly during the late exponential and spore-releasing phases. Because these genes are structurally organized with sleB171 as an integrated operon, these proteins could potentially perform closely coordinated functions during physiological intracellular processes.

Bacillus thuringiensis has been successfully used as a biocontrol agent for many years. However, one of the technical problems in the application of B. thuringiensis-derived biopesticides is the limited persistence of ICPs in the field. This phenomenon is caused by the exposure of ICPs under harsh environmental conditions and related to the fast shift of cells into dormant spores and the release of ICPs under the action of various PGHs. Therefore, identifying various PGHs involved in cell lysis at the stationary phase is crucial for elucidating the release mechanisms of spores and ICPs during cell division. Thus, further studies are recommended to focus on the activities and regulation of various PGHs during the late stationary phase of B. thuringiensis cells to generate the controlled release of spores and ICPs as an approach to promoting field persistence.

In conclusion, we functionally analyzed a cell wall hydrolase-encoding gene, sleB171, which is involved in daughter cell separation during cell division for B. thuringiensis BMB171 cells. The disruption of this gene in host cells resulted in the formation of long cell chains during the vegetative growth phase and delayed the spore-formation and spore-release phases; however, it did not have a significant impact on the eventual release and overall growth of the cells.

Supplementary Data

Supplementary Data is available at ABBS online.

Funding

This work was financially supported by the grants from the National Basic Research Program of China (No. 2013CB127504), the National Natural Science Foundation of China (No. 31270158), and Non-Profit Science and Technology Research Funds of Hubei Province of China (No. 2012DBA10001).