The phosphotransferase system gene ptsI in the endophytic bacterium Bacillus cereus is required for biofilm formation, colonization, and biocontrol against wheat sharp eyespot

Authors


Abstract

Natural resistance of wheat plants to wheat sharp eyespot is inadequate, and new strategies for controlling the disease are required. Biological control is an alternative and attractive way of reducing the use of chemicals in agriculture. In this study, we investigated the biocontrol properties of endophytic bacterium Bacillus cereus strain 0–9, which was isolated from the root systems of healthy wheat varieties. The phosphotransferase system is a major regulator of carbohydrate metabolism in bacteria. Enzyme I is one of the protein components of this system. Specific disruption and complementation of the enzyme I-coding gene ptsI from B. cereus was achieved through homologous recombination. Disruption of ptsI in B. cereus caused a 70% reduction in biofilm formation, a 30.4% decrease in biocontrol efficacy, and a 1000-fold reduction in colonization. The growth of ΔptsI mutant strain on G-tris synthetic medium containing glucose as the exclusive carbon source was also reduced. Wild-type properties could be restored to the ΔptsI mutant strain by ptsI complementation. These results suggested that ptsI may be one of the key genes involved in biofilm formation, colonization, and biocontrol of B. cereus and that B. cereus wild-type strain 0–9 may be an ideal biocontrol agent for controlling wheat sharp eyespot.

Introduction

Bacillus cereus is a Gram-positive, rod-shaped, aerobic, or facultatively anaerobic bacterium that can form endospores (Broussolle et al., 2010). It can be isolated from a wide range of environments, including soil and the rhizosphere of plants (Dunn et al., 2003; Mols & Abee, 2011). There are several reports about B. cereus and its products used as biocontrol agents for the suppression of some diseases occurring in crop plants and in plants kept in aquaculture (Smith et al., 1993; Silo-Suh et al., 1994; Stabb et al., 1994; Lalloo et al., 2010).

Wheat sharp eyespot is a serious disease of wheat (Triticum aestivum L.) in China. Its main causal agent is Rhizoctonia cerealis (Chen et al., 2008; Liu et al., 2009a). Rhizoctonia cerealis can infect stems and sheaths of wheat plants, leading to a block in the transportation of nutrients required for the growth of wheat (Chen et al., 2008). Yield loss and quality reduction caused by this fungus exceeded ¥1 billion per year in China (Chen et al., 2008). Wheat varieties with natural resistance to wheat sharp eyespot pathogens are limited, which has severely hampered progress in breeding for resistance using conventional approaches (Liu et al., 2009a). Chemical control of plant disease has some severe and undesirable environmental and ecological consequences, such as nontarget effects and occurrence of fungicide resistance in pathogen populations (Emmert & Handelsman, 1999). Therefore, it is necessary to develop new strategies for the effective control of plant diseases.

Biological control is an alternative or a supplemental way of reducing the use of synthetic chemicals in agriculture (Postma et al., 2003; Compant et al., 2005) and may provide an option for combating wheat sharp eyespot.

Endophytic bacteria live inside plant tissues for at least part of their life cycle without causing substantial harm to the host and can be isolated from several types of plant tissues including seeds, roots, leaves, and stems (Lodewyck et al., 2002; Hardoim et al., 2008; Vendan et al., 2010). They can adapt to the ecological niche, have beneficial effects on plants, and can promote plant growth and suppress disease (Feng et al., 2006; Ryan et al., 2007; Vendan et al., 2010; Reinhold-Hurek & Hurek, 2011). Therefore, studying the mechanisms involved in establishing and maintaining this relationship is very important. The use of beneficial endophytic bacteria as biocontrol agents is of particular interest in plant disease protection (Reinhold-Hurek & Hurek, 2011; Schilirò et al., 2012).

The phosphotransferase system (PTS) was first described in Escherichia coli as a carbohydrate transport system (Kundig et al., 1964). To date, a PTS has been found in many different classes of bacteria and is a major regulator of carbohydrate metabolism in bacteria (Pflüger-Grau & Görke, 2010). There are two general types of PTS in bacteria. The first is the phosphoenolpyruvate-dependent carbohydrate PTS, which is a multicomponent phosphotransfer cascade that transfers phosphate moieties derived from phosphoenolpyruvate (PEP) from one member of the system to the next in a given order (Houot et al., 2010ab; Pflüger-Grau & Görke, 2010). The second system, known as the nitrogen PTS (PTSNTr), does not transport carbohydrates but exerts regulatory functions in the bacteria (Zimmer et al., 2008; Pflüger-Grau & Görke, 2010).

The E. coli PTS consists of several protein components: enzyme I (EI), histidine-phosphorylatable protein (HPr), and a membranous enzyme II complex (Barabote & Saier, 2005; Pflüger-Grau & Görke, 2010). Enzyme II complex consists of four proteins or protein domains, namely IIA, IIB, IIC, and IID, with each one being specific for one or a few given substrate sugars (Pflüger-Grau & Görke, 2010). There are several reports suggesting that the bacterial PTS plays a major role in sugar transport and phosphorylation, carbon catabolism, and in the regulation of essential physiological processes, such as biofilm formation and colonization (Comas et al., 2008; Fujita, 2009; Houot et al., 2010ab). In contrast to E. coli, much less is known about the regulation of the PTS in B. cereus.

The biocontrol strain B. cereus 0–9 is an endophytic bacterium that was isolated from the root systems of healthy wheat varieties Yumai70 in Kaifeng, China (Liu et al., 2009b). In the current study, we constructed a TnYLB-1 transposon random insertional mutant library of B. cereus strain 0–9 and isolated a mutant with significantly reduced biological control activity. Inverse PCR was used to locate the insertion site, which was identified as the PTS component protein enzyme I-coding gene ptsI. ptsI deletion and complementation mutant strains were constructed. Disruption of ptsI significantly reduced biocontrol efficacy, biofilm formation, and colonization, compared with the wild-type B. cereus strain. The B. cereus ΔptsI mutant strain also demonstrated a reduced ability to grow on a medium with glucose as the sole carbon source. Reintroduction of a functional ptsI into the ΔptsI mutant strain restored the wild-type phenotype. These results demonstrated the important in vivo role played by ptsI during the transfer of the phosphate moiety through the PTS, suggesting that ptsI may be one of the key genes involved in the biocontrol, biofilm formation, and colonization of B. cereus. Based on the results described above, we suggest that the B. cereus wild-type strain 0–9 would be an ideal biocontrol agent for controlling wheat sharp eyespot.

Materials and methods

Strains, plasmids, and culture conditions

Biocontrol strain B. cereus 0–9 was obtained from the China Center for Type Culture Collection (CCTCC No.: M209041) and is the subject of a patent that has been approved in China (Patent No.: 200910064867.2). Bacillus cereus 0–9 was stored in a Luria–Bertani (LB) medium (Sambrook et al., 2001) containing 25% glycerol at −70 °C until needed. Escherichia coli 116 (pir+) (Supporting Information, Table S1) cells were grown in a LB medium for the propagation of plasmids for DNA extraction. Escherichia coli GM2163 (dam−) (Table S1) was grown in a LB medium for the propagation of plasmids without methylation. Rhizoctonia cerealis strain HD-6 (Table S1) was cultured on potato dextrose agar (PDA) for biocontrol assays. Plasmid pMarB, with temperature-sensitive replicon repG+ts and the TnYLB-1 transposon, was used for the construction of random transposition mutants (Breton et al., 2006). Plasmid pMAD, containing ampicillin and erythromycin resistance genes, was used for generating gene inactivation mutants (Arnaud et al., 2004). Plasmid pAD123, containing ampicillin and chloramphenicol resistance genes, was used for gene complementation (Dunn & Handelsman, 1999).

Phenotype and growth curve assay

LB and G-tris synthetic media (Nakata & Halvorson, 1960) were used for colony phenotype analysis and growth curve assays of the different strains. Two-microliter aliquots of bacterial suspension were spotted onto the center of LB or G-tris synthetic medium plates. The plates were incubated at 30 °C in darkness. Three replicates were carried out, and photographs were taken 5 days postinoculation. Two-microliter aliquots of bacterial suspension were inoculated into 5 mL LB or G-tris synthetic media and cultured at 30 °C for 24 h in darkness. The bacterial cells were harvested and adjusted to OD600 = 0.1 in LB or G-tris synthetic media. Three-hundred microliter aliquots of the bacterial suspensions were inoculated into the microchambers, and the OD600 was measured as turbidity per hour using an automated turbidimetric system (Bioscreen C, Type: FP-1100-C, Finland). The experiment was replicated 10 times.

DNA manipulations

Plasmid and genomic DNA were extracted following the standard trizol extraction methods (Sambrook et al., 2001). All of the restriction endonucleases and modifying enzymes were purchased from Takara Biotechnology Company (Dalian, China) and used according to the manufacturer's instructions. PCRs were performed with a Thermal cycler (Applied Biosystems Veriti 96-well thermal cycler, Life technologies, Singapore). The PCR primers (Table S2) were designed according to the ptsI sequence of the B. cereus strain ATCC 14579 (GenBank accession no: AE016877.1). The primer pair ptsI-up-bam-s and ptsI-up-sal-a, with BamHI and SalI ends, respectively, were used to amplify a 1100-bp fragment upstream of ptsI. The primer pair ptsI-d-sal-s and ptsI-d-nco-a, with SalI and NcoI ends, respectively, were used to amplify a 1100-bp fragment downstream of ptsI. The thermal cycler conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 1 min (with a last-cycle hold of 72 °C for 10 min). The two fragments were gel-purified (DNA Gel Extraction Kit; DingGuo, Beijing, China), digested with SalI, and then ligated with T4 DNA ligase to form a new 2200-bp fragment, pstI-AB. This ptsI-AB fragment was digested with BamHI and NcoI and then ligated with T4 DNA ligase into the BamHI/NcoI site of pMAD to generate the ptsI gene deletion vector pMAD-AB.

To construct the ptsI complementation vector, the PCR primer pair ptsIcom-BamHI-S and ptsIcom-HindIII-a, with BamHI and HindIII ends, respectively, were used to amplify the ptsI-coding sequence and its native promoter sequence. The 2200-bp PCR fragment was purified and digested with BamHI and HindIII and then cloned into the BamHI/HindIII site of pAD123 (Dunn & Handelsman, 1999). The resulting complementation vector was designated pAD123-ptsIcom.

Construction of transposon random insertional mutants

The random mutagenesis of B. cereus by the TnYLB-1 transposon was performed according to Breton et al. (2006) with some modifications. The plasmid pMarB was transformed into the B. cereus strain 0–9 protoplasts by electroporation (1700 V, Eporator, Eppendorf, Hamburg, Germany), followed by selection for kanamycin (50 μg mL−1) and erythromycin (5 μg mL−1) resistance. The transformants were inoculated into LB medium supplemented with kanamycin (50 μg mL−1) and erythromycin (5 μg mL−1) and incubated with shaking at 200 r.p.m. for 24 h at 30 °C. The cultures were then spread onto LB agar plates supplemented with kanamycin (50 μg mL−1) and incubated at 46 °C for 10 h. Subsequently, the plates were placed at 30 °C overnight until the transformants appeared. The transformants were picked onto LB agar plates supplemented with kanamycin (50 μg mL−1) and then duplicated on plates containing erythromycin (5 μg mL−1) and incubated at 30 °C overnight. Transformants that were sensitive to erythromycin but not kanamycin contained the correct insert. The transformants were selected with erythromycin and kanamycin three times and then stored with 25% glycerol at −70 °C until needed.

Southern blot analysis

For transposon random insertional mutants, the bacterial genomic DNA (3 μg) was digested with EcoRI, electrophoresed on 0.8% agarose gels, and then transferred to Hybond-N+ membrane (Amersham Biosciences, Amersham, UK). A TnYLB-1 transposon fragment (1.5 kb) containing the kanamycin resistance gene, digested from pMarB with PstI, was labeled with a Random Prime DNA Labeling system (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche, Mannheim, Germany) and used as a hybridization probe. The labeling efficiency was determined according to the manufacturer's instruction. Southern blotting was performed following a standard protocol described by the manufacturer (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche).

For the ptsI gene deletion and complementation strains, the Southern blotting was performed according to the protocol described above with an exception. The hybridization probe was PCR-amplified using primer pair ptsIRTP1/ptsIRTP2 (Table S2) with genomic DNA of B. cereus strain 0–9 as a template.

Inverse PCR

Inverse PCR, described previously by Ochman et al. (1988), was used to determine the insert location of the TnYLB-1 transposon in the random insertional mutants. For inverse PCR detection, genomic DNA from B. cereus TnYLB-1 random insertional mutants was extracted according to a standard trizol extraction protocol (Sambrook et al., 2001). Genomic DNA was digested with TaqI and then self-ligated with T4 DNA ligase (Takara Biotechnology Company). The resulting ligation product was used as substrate for PCR amplification. The thermal cycler conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 50 s (with a last-cycle hold of 10 min). The PCR primers used in this reaction were olPCR1 and olPCR2 (Table S2). The resulting amplicons were purified (DNA Gel Extraction Kit, DingGuo) and sequenced. The DNA sequences were used for database analysis using the blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the insertion site of the TnYLB-1 transposon in the B. cereus genome.

Construction of a gene deletion mutant

The ptsI gene deletion mutant strain was constructed according to Arnaud et al. (2004) with some modifications. The pMAD-AB vector was transformed into E. coli 116-competent cells by electroporation (1700 V, Eporator, Eppendorf) for propagation of the plasmid. pMAD-AB was extracted and identified with restriction endonucleases. Subsequently, the plasmid was transformed into E. coli GM2163 and also extracted and identified with restriction endonucleases. The pMAD-AB plasmid from E. coli GM2163 was transformed into B. cereus strain 0–9-competent cells by electroporation (1700 V, Eporator, Eppendorf) and cultured on tryptic soy agar (TSA; BD Diagnostics, Sparks, MD) plates supplemented with erythromycin (3 μg mL−1) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal, 100 μg mL−1) at 30 °C overnight. One blue colony was inoculated into 50 mL of TSB medium (BD Diagnostics) supplemented with erythromycin (5 μg mL−1) and incubated at 30 °C at 200 r.p.m. overnight. Five hundred microliters of the culture was inoculated into 50 mL of TSB medium in a flask. The flask was incubated at 30 °C at 200 r.p.m. until the optical density of the culture reached 0.01 at 600 nm, and then incubated with shaking for 2 h at 30 °C at 200 r.p.m. followed by 6 h at 42 °C. One hundred microliters of this culture was plated on TSA agar plates containing erythromycin (3 μg mL−1) and X-gal (100 μg mL−1) and incubated at 42 °C for 48 h. One blue colony was inoculated into 50 mL of TSB medium supplemented with erythromycin (5 μg mL−1) and incubated at 30 °C and 200 r.p.m. overnight. One hundred microliters of this culture was plated on TSA agar plates containing X-gal (100 μg mL−1) and incubated at 30 °C overnight. The white colonies were isolated on the same medium at 30 °C and verified for erythromycin sensitivity. To confirm the gene deletion, chromosomal DNA was extracted from two candidate clones and used in PCR with the ptsI-up-bam-s and ptsI-d-nco-a primer pair.

Complementation of ptsI

The ptsI gene complementation vector pAD123-ptsIcom from E. coli GM2163 was transformed into competent B. cereus ΔptsI cells by electroporation (1700 V, Eporator, Eppendorf). Transformants were cultured on TSA plates supplemented with chloramphenicol (10 μg mL−1) at 30 °C overnight. The plasmid was extracted and identified by PCR using the primer pair ptsIcom-BamHI-S/ptsIcom-HindIII-a.

RT-PCR assay

Total RNA was extracted from 5 mL of the log-phase bacterial using a bacterial RNA extraction kit (E. Z. N. A. Bacterial RNA Kit, OMEGA Bio-Tek, Inc., Beijing, China) and treated with RNase-free DNase I (Takara). The bacterial RNA was reverse-transcribed with PrimeScript Reverse Transcriptase (Takara) and the ptsI gene-specific primer ptsIRTP1/ptsIRTP2 and 16S rRNA gene-specific primer 16SrRNARTP1/16SrRNARTP2 (Table S2). The synthesized cDNAs were subjected to RT-PCR with ptsI gene-specific primers ptsIRTP1/ptsIRTP2. The 16S rRNA gene (GenBank accession no: NC_004722.1) of B. cereus was used as an internal control during RT-PCR.

B. cereus biofilm assay

Solid surface-associated biofilm formation was estimated by the crystal violet (CV) staining method, with some modifications (Morikawa et al., 2006). A single colony of B. cereus strain 0–9 and its transformants were inoculated into 2 mL of LB medium and incubated at 30 °C overnight. Twenty microliters of the overnight culture was inoculated into 2 mL of modified MSgg medium (Branda et al., 2004) in glass culture tubes with a diameter of 0.8 cm. The tubes were incubated upright at 22 °C for 5 days before surface pellicles and cultures were carefully removed from the tubes. The remaining cells and matrices in each tube were stained with 2.5 mL of 0.1% (w/v) CV solution for 20 min at room temperature. After washing three times with distilled water, the CV attached to the biofilm was solubilized in 2.5 mL 10% (w/v) SDS. Three hundred microliters of the solution was quantified by measuring its absorbance at 570 nm. Ten replicates were carried out for each strain.

Colonization assay

The commercially available wheat cultivar Aikang58 was used for colonization assays. A single colony of B. cereus wild-type strain 0–9 and its transformants were inoculated into 50 mL of LB medium and incubated with shaking for 16 h at 30 °C and 200 r.p.m. The bacterial cells were harvested by centrifugation at 2350 g for 10 min. The precipitates were adjusted to OD600 = 0.8 in 40 mL of 10 mM MgSO4. The germinated wheat seeds were soaked in the bacterial cultures for 1 h and then vacuumized for 10 min. Five wheat seeds treated with each of the different bacterial strains described above were planted in sand mixed with vermiculite (v : v = 3 : 1). Five replicates were used for each bacterial strain. The planted seeds were placed at 22 °C with continuous fluorescent lighting. At 7, 14, and 20 days postinoculation, 0.3 g fresh weight of the underground wheat seedlings was disrupted in a mortar and pestle. The suspensions were diluted by 106 times with appropriate amounts of 10 mM MgSO4, and ten microliters of the diluted suspension was plated on LB agar plates and then incubated at 30 °C for 24 h. Bacterial colony-forming units on each plate were counted.

Biocontrol assay

Rhizoctonia cerealis strain HD-6 was inoculated into the autoclaved wheat kernels in a conical flask and incubated at 28 °C in darkness for 2 weeks. The B. cereus wild-type strain 0–9, TnYLB-1 random insertional mutant strain, ΔptsI mutant strain, and the ptsI-complemented strain were inoculated in LB medium and incubated at 30 °C overnight. The bacterial cells were harvested by centrifugation at 2350 g for 10 min and adjusted to a final concentration of 2 × 109 mL−1 with LB broth. The kernels with abundant mycelia were placed on the autoclaved soil–sand mixture (v : v = 1 : 1) in a 10-cm-diameter plastic pot. Germinated Aikang58 wheat seeds were also planted on the soil–sand mixture neighboring to the kernels. Ten kernels and five seeds were planted in one pot and then covered with a 2.5 cm deep of the soil–sand mixture. Five replicate pots were used per bacterial strain. Ten milliliters of bacterial suspension of each strain (2 × 109 mL−1) was irrigated into the soil–sand mixture of each pot and the pots were incubated at 20 °C under conditions of a 12-h photoperiod and 90% relative humidity. Twenty days later, the wheat seedlings were washed and the sharp eyespot lesions on the base of wheat culms were evaluated. The disease index and the incidence of the disease were measured according to Lipps & Herr (1982). The experiment was replicated three times.

Results

TnYLB-1 random insertional mutant has reduced ability to form a biofilm

We constructed a TnYLB-1 random insertion mutant library containing 661 mutants and selected 10 transformants from the mutant library that showed a reduced ability to form biofilms (Supporting Information, Fig. S1). As shown in Fig. S1, mutant strain 5 showed a 70% reduction in biofilm formation (< 0.01) compared with the wild-type strain 0–9.

Southern blot analysis of the TnYLB-1 random insertional mutant strains

To determine the copy number of the TnYLB-1 transposon inserted into the genome of B. cereus, genomic DNA from the 10 mutant strains with reduced biofilm formation ability was used for the Southern blot analysis. A TnYLB-1 transposon fragment (1.5 kb) containing the kanamycin resistance gene was used as DNA probe. As shown in Fig. S2, the 10 selected mutant strains had different copy numbers and insertion locations. Mutant strain 5 had only one TnYLB-1 insertion. It was defective in forming a biofilm and had reduced biocontrol efficiency (see below) and was therefore selected for further investigation.

Inverse PCR

To identify the insertion site of the TnYLB-1 transposon in mutant strain 5, the genomic DNA of the mutant strain was extracted and used for inverse PCR. The 300-bp PCR product was sequenced and then subjected to blast analysis in the NCBI database. Results indicated that the inverse PCR fragment had 99% identity to the phosphotransferase protein-coding gene ptsI of B. cereus strain ATCC 14579. These results suggested that the TnYLB-1 transposon randomly inserted into the ptsI-coding sequence of B. cereus strain 0–9 (Fig. 1b). The ptsI gene of B. cereus strain 0–9 was found to have a close phylogenetic relationship with other ptsI genes of different B. cereus strains (Fig. S3).

Figure 1.

Construction of the ptsI gene deletion and complementation mutants and the PCR, RT-PCR, and Southern blot analysis. (a) Physical map of pMAD-AB and the construction of ΔptsI mutant strain. A double cross-over event occurred between the pMAD-AB plasmid (upside) and the ptsI gene (middle part), resulting in the generation of the ΔptsI mutant (bottom). The arrowheads indicate the location of the PCR primers used in this study. The A and B fragments (black boxes) represent the upstream and downstream sequence of the ptsI, respectively. The gray box represents the coding sequence of the ptsI gene. (b) Construct of the TnYLB-1 transposon insertional mutant strain. The transposon inserted into the ptsI gene-coding sequence. The insertion site located between the primer P9 and P10. (c) PCR analysis of the ΔptsI mutant strains. M: DNA molecular weight marker. Lane 1: PCR products (1.7 kb) amplified using the primers ptsI-28a-BamHI-s/ptsI-28a-XhoI-a and the wild-type strain 0–9 genomic DNA as template. Lane 2: PCR products (4 kb) amplified using the primers ptsI-up-bam-s/ptsI-d-nco-a and the wild-type strain 0–9 genomic DNA as template. Lane 3: PCR products (2.2 kb) amplified using the primers ptsI-up-bam-s/ptsI-d-nco-a and the ΔptsI gene deletion mutant strain genomic DNA as template. Lane 4: PCR products (2.2 kb) amplified using the primers ptsIcom-BamHI-S/ptsIcom-HindIII-a and the wild-type strain 0–9 genomic DNA as the template. The PCR fragment was used for ptsI complementation. Lane 5: PCR products (3.9 kb) amplified using the primers ptsIcom-BamHI-S/ptsIcom-HindIII-a and TnYLB-1 transposon insertional mutant strain 5 genomic DNA as the template. The PCR fragment contained the upstream and downstream sequence of ptsI and the TnYLB-1 transposon sequence. (d) RT-PCR analysis of the ΔptsI mutant strains for ptsI gene expression with the gene-specific primers ptsIRTP1 and ptsIRTP2. M: DNA molecular weight marker. Lane 1: PCR product with the cDNA of wild-type strain 0–9 as template. Lane 2: PCR product with the cDNA of TnYLB-1 transposon insertional mutant strain as template. Lane 3: PCR product with the cDNA of ΔptsI mutant strain as template. Lane 4: PCR product with the cDNA of ptsI-complemented strain as template. Housekeeping gene 16S rRNA was used as an internal control. The locations of the primers are indicated in Fig. 1a. (e) Southern blot analysis of the ΔptsI mutant strains. Lane 1: wild-type strain 0–9. Lane 2: TnYLB-1 transposon insertional mutant strain. Lanes 3: ΔptsI mutant strain. Lane 4: ptsI-complemented strain. The probe was PCR-amplified using primer pair ptsIRTP1/ptsIRTP2 (Table S2) with 0–9 genomic DNA as a template. The locations of the primers are indicated in Fig. 1a.

ptsI deletion and complementation in B. cereus strain 0–9

The pMAD-AB vector was transformed into the B. cereus strain 0–9 and ptsI was deleted through a double cross-over event (Fig. 1a). Twenty-one transformants were obtained from one experiment. The transformants were analyzed by PCR, RT-PCR, and Southern blot experiments, respectively. As shown in Fig. 1c, the ΔptsI transformant and the wild-type strain generated PCR products of the expected 2.2- and 4-kb fragments, respectively. RT-PCR primers ptsIRTP1/ptsIRTP2 spanned a fragment of the ptsI gene-coding sequence (Fig. 1a; Table S2) and amplified a fragment of 484 bp from strain 0–9 and ptsI-complemented strain (see below; Fig. 1d). In contrast, no such RT-PCR products were seen from the TnYLB-1 insertional mutant strain and the ΔptsI mutant strain (Fig. 1d), indicating that ptsI transcripts were absent from these strains. Southern blot analyses also confirmed the absence of the ptsI gene in the ΔptsI mutant strain (Fig. 1e). These results indicated that the ΔptsI strain had complete deletion of ptsI.

The pAD123-ptsIcom vector was transformed into the ptsI gene deletion mutant strain and expressed, resulting in 14 ptsI complementation mutant strains. The ptsI-complemented strain showed the expected 2.2-kb PCR fragment (Fig. 1c).

Phenotypes of ΔptsI and ptsI-complemented strains

The ΔptsI mutant strain and TnYLB-1 insertional mutant strain formed smaller colonies than the 0–9 wild-type and the ptsI-complemented strain when grown on a G-tris synthetic medium using glucose as the sole carbon source, but was indistinguishable from the other strains when grown on an LB medium (Fig. 2a). This decreased growth was also confirmed in in vitro growth assays (Fig. 2b). On the G-tris synthetic medium using glucose as the sole carbon source, the ΔptsI mutant strain had a significantly lower growth rate than the 0–9 wild-type strain, indicating that deletion of ptsI suppresses glucose metabolism. In the LB medium, the ΔptsI strain produced a growth curve similar to that of the 0–9 wild-type strain (Fig. 2c).

Figure 2.

Phenotype and growth curves of wild-type strain 0–9, the TnYLB-1 transposon insertional mutant strain (ptsI-T), the ΔptsI mutant strain (ΔptsI), and the ptsI-complemented strain (ptsI-C). (a) Phenotypes of the mutant strains. G-tris synthetic medium and LB medium were used in this assay. Two microliters of bacterial suspension (OD600 = 0.4) was inoculated into G-tris or LB plates and then placed at 30 °C in darkness. Three replicates were carried out and representative photographs were taken 5 days postinoculation. (b) The growth curves of different strains on G-tris synthetic medium. (c) The growth curves of different strains on LB medium. The growth of the different strains was measured with an automated turbidimetric system (Bioscreen C, Finland). The experiment was replicated 10 times.

Biofilm assay

The B. cereus wild-type strain 0–9, TnYLB-1 insertional mutant strain, ΔptsI mutant strain, and the ptsI-complemented strain were used for the biofilm formation assay. The ΔptsI mutant strain produced significantly less biofilm than the wild-type strain 0–9 and the ptsI-complemented mutant (Fig. 3). While the ability of biofilm formation of ΔptsI was reduced by 70% compared with that of the wild-type strain, the ptsI-complemented strain produced a similar amount of biofilm as compared with the WT strain. These results suggested that disruption of ptsI reduces biofilm formation ability of B. cereus strain 0–9.

Figure 3.

Comparison of biofilm formation of wild-type strain 0–9, the ptsI transposon insertional mutant strain (ptsI-T), the ΔptsI mutant strain (ΔptsI), and the ptsI-complemented strain (ptsI-C). Biofilm formation was estimated by the crystal violet (CV) staining method. Data are expressed as the absorbance at 570 nm. The data are the average ± standard error from 10 replicates. (a) Biofilm formation of the ΔptsI mutant strains. The photographs were taken after 5 days of incubation. (b) Data analysis of biofilm formation ability of the ΔptsI mutant strains. Different letters represent a significant difference at < 0.01.

Colonization assay

Bacillus cereus wild-type strain 0–9 and its transformants were analyzed for their colonization competence. The ΔptsI mutant and the TnYLB-1 insertional mutant showed a 1000-fold reduction in colonization of wheat roots, as compared with the wild-type strain. Colonization by the ptsI-complemented strain was similar to the wild-type strain (Table 1). This result suggested that disruption of ptsI affects the ability to colonize wheat roots.

Table 1. Colonization assay of B. cereus wild-type strain 0–9, TnYLB-1 insertional mutant strain, ΔptsI mutant strain 5, and ptsI-complemented strain on wheat root
StrainGenotypeColonization ability (104 CFU g−1 roots)
7 days14 days20 days
  1. Data are expressed as bacterial CFU (104) per gram of roots. The data are the average ± standard error from five replicates. Different letters represent a significant difference at P < 0.01.

0–9Wild type54.3 ± 2.61a30.0 ± 7.00a1.70 ± 0.26a
ptsI-TTnYLB-1 transposon insertional mutant0.09 ± 0.03b0.02 ± 0.004b0.008 ± 0.001b
ΔptsIΔptsI mutant0.07 ± 0.01b0.01 ± 0.002b0.004 ± 0.002b
ptsI-CptsI complemented 42.7 ± 12.7a24.0 ± 1.53a1.20 ± 0.17a

Biocontrol assay

The B. cereus strains described above were subjected to biocontrol assay. The disease incidence of wheat inoculated with R. cerealis strain HD-6 was 83.4. In plants simultaneously inoculated with R. cerealis and B. cereus, the disease incidence was reduced by 31.4%. Wheat plants inoculated with R. cerealis and the ΔptsI mutant showed disease incidence comparable to those plants inoculated with R. cerealis alone (Table 2). Comparable data were found on the basis of the disease index (Table 2). These results suggest that B. cereus has a strong biocontrol effect against wheat sharp eyespot and that disruption of ptsI has a profound effect on bacterial growth and development, leading to a reduced biocontrol ability.

Table 2. Biocontrol assay of B. cereus wild-type strain 0–9, the TnYLB-1 insertional mutant, the ΔptsI mutant strain, and the ptsI-complemented strain against wheat sharp eyespot
StrainGenotypeDisease indexDisease incidence
  1. The data are an average ± standard error from three replicate experiments. Different letters represent a significant difference at < 0.01.

  2. a

    Wheat seeds that were inoculated with R. cerealis HD-6 without B. cereus suspension as a control.

CKa 60.1 ± 0.76a83.4 ± 2.82a
0–9Wild type42.4 ± 1.51b57.2 ± 2.87b
ptsI-TTnYLB-1 insertional mutant57.6 ± 1.63a79.8 ± 1.89a
ΔptsIΔptsI mutant55.3 ± 2.53a79.5 ± 2.86a
ptsI-CptsI complemented51.4 ± 1.87ab68.5 ± 2.56ab

Discussion

Specific disruption and complementation of ptsI from B. cereus was achieved through homologous recombination. Disruption of the ptsI gene caused a significant reduction in biofilm formation and colonization, as well as affecting bacterial growth on the G-tris synthetic medium with glucose as the sole carbon source, as well as biocontrol efficacy. The changes in the ΔptsI mutant strain were partially restored by ptsI complementation. These results suggest that ptsI may be one of the key genes involved in biofilm formation, colonization, and biocontrol of B. cereus and that the B. cereus wild-type strain 0–9 is an ideal candidate for development as a biocontrol agent for controlling wheat sharp eyespot.

Biocontrol agents can act through several mechanisms, including production of metabolites, production of antibiotics against the pathogen, competition for nutrients, and induction of plant resistance (Haggag, 2010). Pseudomonas chlororaphis MA 342 is a potent biocontrol agent and it can colonize cereal plant roots where it then produces fungistatic compounds against the pathogens (Tombolini et al., 1999). Bacillus cereus UW85 can produce two antibiotics to suppress the damping-off of alfalfa caused by Phytophthora medicaginis and the fruit rot of cucumber caused by Pythium aphanidermatum (Smith et al., 1993; Silo-Suh et al., 1994). In this study, we used the cultures and culture filtrates of B. cereus strain 0–9 and its transformants to carry out an inhibition assay against the R. cerealis strain HD-6, but unexpectedly they all did not have a significant suppression to the fungal pathogen (Fig. S4). This result indicated that the mechanism of biocontrol effect of B. cereus strain 0–9 did not rely on the secretion of fungistatic compound.

The ability of biocontrol agents to control plant disease is dependent on the colonization of plant surfaces (Haggag, 2010). The B. cereus ΔptsI mutant strain showed a significant reduction in the ability to colonize the host plant by three orders of magnitude, as compared with that of the wild type (Table 1).

Biofilms are surface-attached communities of bacteria embedded in an extracellular matrix comprising exopolysaccharide, proteins, and DNA (Morikawa et al., 2006; Absalon et al., 2012). Biofilm formation can mediate protection from a wide range of environmental challenges (Hall-Stoodley et al., 2004). There are several reports about the role of bacterial biofilms in the biocontrol of plant diseases (Bais et al., 2004; Haggag, 2010; Chen et al., 2013). Bacillus subtilis strain 6051 can form a stable and extensive biofilm to protect Arabidopsis against attack by the bacterial pathogen Pseudomonas syringae (Bais et al., 2004). Chen et al. (2013) isolated and screened six B. subtilis strains from China that exhibited at least 50.0% biocontrol efficacy on tomato against the pathogen Ralstonia solanacearum under greenhouse conditions. These B. subtilis strains formed robust biofilms on tomato roots and exhibited strong antagonistic activities against other various plant pathogens. In the current study, the B. cereus strain 0–9 that can form robust biofilms has a profound biocontrol efficacy against wheat sharp eyespot. The ΔptsI mutant exhibited a 70.0% reduction in biofilm volume compared with that of the wild-type strain, along with a 30.4% increase in disease index compared with the wild type (Fig. 3 and Table 2). These results suggest that ptsI contributes substantially to the formation of the biofilm matrix in B. cereus 0–9 and deletion of this gene leads to a reduced biocontrol efficacy.

In our study, we also used the B. cereus 0–9 and its transformants to perform the biocontrol assay against other plant diseases, such as the take-all of wheat caused by Gaeumannomyces graminis var tritici and the sheath blight of rice caused by Rhizoctonia solani AG-1. The results indicated that the B. cereus 0–9 and its transformants have no biocontrol efficacy to these fungal pathogens (data not shows).

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31300001, 30971952, 30771435, 31200069) and the China Postdoctoral Science Foundation (2011M501178). The authors would also like to thank Dr Kazuya Morikawa (University of Tsukuba, Japan) for generously providing plasmid pMAD as a gift.

Authors' contribution

Y.-B.X. and M.C. contributed equally to this work.

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