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Research Article
18 July 2012

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy


The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T 1), early sporulation (T 2), and late sporulation (T 3) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.


Bacillus thuringiensis is characterized by the production of a parasporal body during sporulation, which contains one or more Cry and/or Cyt proteins (also known as δ-endotoxins) in crystalline form (17, 42). Many B. thuringiensis subspecies are used as bacterial insecticides and sources of genes for the recombinant bacteria and B. thuringiensis strains applied for insecticidal products (5, 31). Since the 1980s, substantial progress has been made in the development and production of new genetically engineered B. thuringiensis insecticides, especially in the transformation of insecticidal crystal proteins (ICPs), due to the ever-increasing research on the B. thuringiensis protein production mechanisms and genetic structure. For B. thuringiensis, many researchers have found great difficulty in achieving marked improvements in ICP only by traditional fermentation and mutagenesis treatment, which hinders the practical applications of B. thuringiensis. The challenge is probably rooted in the fact that except for the closely synergistic effect with spore formation (2), ICPs are synthesized via multiple intracellular reactions, which are further complicated by various factors, including cofactor balance and regulator circuitry at different levels of cell metabolism. Although the expression and regulation of crystal genes have been widely studied (44), the mechanisms of crystal formation and assembly in B. thuringiensis remain unclear (40). Hence, an in-depth study on the synthesis mechanisms of B. thuringiensis parasporal crystals and spores under different physiologically relevant conditions at the proteomic level is highly significant. To date, most B. thuringiensis proteomic studies have mainly focused on parasporal crystal purification and identification (7, 52), spore composition and function (13, 22), classification of Bacillus strains (16), and so on. There has been no report on the large-scale characterization of the B. thuringiensis proteome, which is becoming one of the international hot spots of research.
Traditionally, the most widely used proteomic method for complex protein mixtures prior to mass spectrometry (MS) analysis has been two-dimensional polyacrylamide gel electrophoresis (2D PAGE), followed by enzymatic digestion of the separated protein spots (32). 2D PAGE has emerged as a valuable technique for proteomic studies of the model organism Bacillus subtilis (4) and the Bacillus cereus group of bacteria, including Bacillus anthracis (11), B. cereus (23), and B. thuringiensis (9). Despite the recent progress of 2D PAGE in terms of reproducibility, automation, and quantification (37), the technique still suffers from major drawbacks related to the detection of low-abundance proteins, hydrophobic proteins, and proteins with extreme sizes and charges (28). 2D PAGE is also unlikely to meet the high-throughput demands of the global profiles of protein expression. To overcome these problems with 2D PAGE gels, multidimensional chromatography appears to be a promising alternative. This method provides high peak capacities and allows for the separation of complex digests or complex intact protein mixtures. By this technique, digested peptides can be separated effectively by high-performance liquid chromatography (HPLC), and the isolated peptides can then be directly analyzed by MS. Compared with 2D PAGE, 2D liquid chromatography-tandem MS (LC-MS/MS) is more flexible. It allows for the combination of different separation techniques (36, 48). Samples can be tagged and modified before, between steps, or after a single separation step, and the sequence of multiple runs can be easily automated. 2D chromatography can often be accomplished by either an online or off-line approach, both of which have advantages and disadvantages (39). The most commonly used 2D chromatography technique is the combination of a strong cation-exchange (SCX) column and reversed-phase (RP) separation coupled with a tandem mass spectrometer. This new configuration coupled with high-resolution ion trap MS and an automated data processing system can be a fully integrated analytical platform for non-gel-based large-scale protein identifications. The advantage of this MS method is that a single peptide ion can be isolated and sequenced in the presence of many other codetected peptides. This feature helps circumvent problems with other typical one-dimensional (1D) LC-MS/MS techniques by simplifying the separation pattern and reducing the number of peptides eluted in one fraction (47).
In the present study, an online 2D nano-LC-MS/MS method was used to identify the proteome of a new B. thuringiensis subsp. kurstaki strain, 4.0718, in a fermentation medium at three distinct development stages—namely, the middle vegetative, early sporulation, and late sporulation phases. After trypsin digestion, the separated peptides were directly analyzed by electrospray ionization-MS/MS. The acquired MS and MS/MS spectra were searched against an in-house database using the SEQUEST search engine for protein identification. The Kyoto Encyclopedia of Genes and Genome (KEGG) pathway and Biocyc collection of Pathway/Genome Databases analyses were performed to investigate the functions of the identified proteins. A number of bioactive factors in B. thuringiensis, including the Cry protoxin, were identified. Western blot analysis was used to confirm the expression of two identified proteins related to the spore and crystal syntheses during the three growth phases of B. thuringiensis. The results lay a significant theoretical foundation for the methodology of analyzing the B. thuringiensis proteome. Consequently, we created a database of protein expression profiles for the B. thuringiensis developmental phases that may facilitate further analyses of the regulation of these complex events.


Strain cultivation and sample preparation.

The wild-type B. thuringiensis strain 4.0718 (CCTCC no. M200016) was isolated from soil in south China. This strain can produce two types of parasporal crystals, namely, bipyramidal and cuboidal (14). After being activated in LB solid medium containing tryptone (10.0 g/liter), yeast extract (5 g/liter), NaCl (10 g/liter), and agar (1.5 g/liter) at 30°C for 12 h, strain 4.0718 was transferred into a 250-ml baffled flask containing 20 ml of fermentation medium. The flask was placed on a rotary shaker, which was operated at 30°C and 220 rpm. The fermentation medium (pH 7.3) contained beef extract (5.0 g/liter), NaCl (2.0 g/liter), MnSO4 (0.05 g/liter), tryptone (10.0 g/liter), MgSO4 · 7H2O (0.3 g/liter), glucose (3.0 g/liter), and K2HPO4 (0.3 g/liter). Two independent series of cultures were prepared and processed (biological replicates) with the same batch of seed and developed simultaneously in the same incubator. Cells were collected every 2 h for microscopic examination and growth curve measurement. The morphology of the B. thuringiensis cells and spores was observed under a phase-contrast microscope (Olympus, Japan) at ×1,000 magnification to confirm the commencement of spore and crystal formation.

Extraction of whole-cell proteins.

Cells harvested at different time points (10, 20, and 32 h) were washed three times with a suspension in previously chilled phosphate-buffered saline (10 mM [pH 7.8]) and placed in a prechilled pestle. Liquid nitrogen was scooped on top of the cells to flash freeze them. The cells were ground into powder using a mortar and pestle. The cell powder (0.2 g) was suspended in 0.8 ml of lysis buffer containing 8 M urea, 4% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonic acid, 40 mM Tris, 65 mM dithiothreitol (DTT), and 5 μl of a protease inhibitor cocktail in a sterilized Eppendorf tube at 4°C for 1 h. The suspension was centrifuged at 4°C and 13,200 rpm for 30 min. The supernatant was precipitated overnight by adding 6 volumes of ice-cold acetone (10% trichloroacetic acid) at −20°C. After centrifugation and lyophilization to dryness, the pellets were stored at −40°C until use.

Protein digestion.

The pellets were dissolved in 0.5 ml of a reducing solution (6 M guanidine hydrochloride and 100 mM Tris [pH 8.3]). Protein concentrations were determined by a 2-D Quant kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's specification. About 300 μg of protein was adjusted to 3 μg/μl. The solution was reduced at 37°C by adding 1 μl of 1 M DTT for 2.5 h and then alkylated by adding 5 μl of 1 M iodoacetamide in the dark at room temperature for 40 min. Afterwards, the solution was precipitated using 6 volumes of ice-cold acetonitrile (ACN)-acetone-acetic acid (50:50:0.1 [vol/vol/vol]) for more than 12 h. The pellets were then washed three times with ice-cold pure alcohol, resuspended in 100 μl of 50 mM NH4HCO3, and incubated with trypsin according to a trypsin/protein ratio of 1:100 (wt/wt) at 37°C for 4 h by shaking. Trypsin was again added according to a trypsin/protein ratio of 1:50 (wt/wt) at 37°C overnight. Trypsin activity was quenched using formic acid (FA). The mixed digests were further ultrafiltered by 10-kDa Microcon flitration device (Sartorius, Germany) to remove the large molecules and were lyophilized to dryness.

2D nano-LC-MS/MS analysis.

All samples were analyzed by nano-LC-MS/MS using an LTQ XL mass spectrometer (ThermoFisher, San Jose, CA) equipped with two Finnigan quaternary pumps (LC Packings, San Jose, CA) and an autosampler (LC Packings, San Jose, CA) equipped with a two-position, 10-port valve having a 25-μl sample loop. The mass spectrometer was equipped with a homemade nano-ionization source.
The principle of the 2D nano-HPLC and valve configuration is illustrated in Fig. S1 in the supplemental material). For the first dimension, 20 μl of redissolved digest (∼50 μg of total peptides) in the 100-μl lined pipe set of the autosampler tray was injected into the sample loop and pumped into the strong cation-exchange column (BioBasic SCX; 0.32 mm by 100 mm, 5 μm) through the sample pump. The SCX column inlet was directly connected to the two-position, six-port valve located in the autosampler. The SCX column outlet was connected to the two-position, 10-port valve located in the mass spectrometer. The 10 ports contained two parallel enrichment columns (Zorbax; 0.3 mm by 5 mm, 5 μm) that were used to trap the peptides eluted from the SCX column by injection of solution plugs with increasing concentrations. After sample injection, the unbinding peptides were trapped in enrichment column 1, and the flow (without ammonium chloride) was directed to the waste, which enabled desalting. At the same time, the mobile phase (2% ACN, 0.1% formic acid [FA]) was pumped into enrichment column 2 by the MS pump, and the flow was directed to a reversed-phase column (BioBasic-C18; 0.1 mm by 150 mm, 5 μm). The outlet of the column was directly connected to the sprayer needle (PicoTip emitter silica tip; New Objective).
After valve switching during a new run, 25 mM ammonium chloride (4% ACN and 0.1% FA) was injected into the sample loop and pumped into the SCX column by the sample pump. The eluted peptides from the SCX column were trapped in enrichment column 2. The mobile phase was 2% ACN and 0.1% FA. At the same time, the peptides trapped in enrichment column 1 were subjected to RP separation followed by MS/MS analysis. The valve was then switched again for the next run. Thus, the eluted peptides were trapped rotatorily in two enrichment columns by a 12-step-elution from the SCX column, and the peptides eluted from the previous run were further separated by an RP column, followed by MS/MS analysis. The salt steps used were 25, 50, 75, 100, 125, 150, 175, 200, 300, 500, and 1,000 mM ammonium chloride (4% ACN and 0.1% FA). The reversed-phase elution gradient procedure was 1 min in 100% buffer A (5% [vol/vol] ACN and 0.1% [vol/vol] FA in water), followed by a 70-min gradient in 65% buffer B (0.1% [vol/vol] FA in ACN), 20-min gradient in 80% buffer B, and 5 min in 80% buffer B, a 1-min gradient in 100% buffer A, and a 12-min reequilibration in 100% buffer A. The flow rate for the reversed-phase separation was maintained at 200 nl/min after splitting.
The peptides eluted from the sprayer needle were electrosprayed directly into the orifice of the mass spectrometer in the positive mode using collision-induced dissociation at a normalize collision energy of 35%. The LTQ mass spectrometer was operated using the “Instrument Method” of the Xcaliber software (Thermo Finnigan). The electrospray voltage was 2.0 kV. The heated capillary was set to 180°C. The capillary voltage was set to 4.5 kV. MS/MS data were acquired in the data-dependent scan mode in which the instrument cycled between the full MS scan (m/z 300 to 2,000) and the intervening MS/MS scans on the 10 most intense ions occurring in the MS scan. The number of ions stored in the ion trap was regulated by automatic gain control. Dynamic exclusion was set at a repeat count of 2, repeat duration of 30 s, and exclusive duration of 90 s.

In-house database search by SEQUEST.

An in-house database was constructed with the protein sequences downloaded from the Uniprot Knowledgebase (Swiss-Prot plus TrEMBL) protein database ( as a FASTA-formatted sequence that included all B. thuringiensis subspecies, which contained 116,723 entries.
The 13 raw files combined for multiple search submission from each sample using Proteome Discoverer Daemon 1.1 were searched against the in-house database on a local server using the SEQUEST search engine (a part of the Proteome Discoverer 1.1 software package). The search parameters were as follows. (i) The mass tolerances of the precursor and fragmentation ions were set to 1.5 and 1.0 Da, respectively. (ii) Two missing cleavage sites were allowed. (iii) Only b and y fragment ions were taken into account. (iv) Static (carbamidomethyl) modification on cysteine and variable modifications, such as oxidation, on methionine were set for all searches. (v) The target-decoy database was also set to satisfy an FDR of ≤0.01 (where FDR is the number of validated decoy entries divided by the number of the total entries multiplied by 100). The other SEQUEST results were kept as default. The target-decoy database was a combination of the original (target) and reversed (decoy) databases. The raw data were searched against the target-decoy database under the same parameters as above. The FDR of the final results was evaluated using the data from the target-decoy database search (33).The search results were further validated manually. Given the similarities among the proteins of the different B. thuringiensis subspecies, when multiple proteins from different subspecies shared the same peptides, they were assigned to a single protein entry to reduce the redundancy of identified proteins. Only the protein that scored the highest was accepted, and the others were omitted. If these homologous proteins were from the same B. thuringiensis subspecies (for example, the isoforms generated by alternative splicing), all of them were reported in the final protein list.

Bioinformatic analysis.

Proteins were classified into functional categories according to their annotated functions in the Uniprot Knowledgebase as well as by homology/functions according to the BioCyc ( and KEGG ( metabolic pathway databases. Protein sequences with unknown functions were subjected to BLASTP query against the UniProt Knowledgebase ( The identified peptides were aligned with the BLAST result, which has been annotated using ClustalW ( to identify protein signatures. The theoretical pI and molecular mass values based on the primary amino acid sequence of the identified proteins were calculated using the Expert Protein Analysis System website (

Validation of selected identified proteins by Western blot analysis.

To validate further the expression of the proteins in the three phases by 2D LC-MS/MS, we examined the existence of two proteins of interest (CotJc and glutamine synthetase) by Western blot analysis. These proteins were chosen because of their crucial roles in the synthesis of spores and crystals. The alterations in protein abundance were derived from the 2D PAGE-MS in our laboratory.
Two genes (cotJc and glnA) that encode the two proteins mentioned above were cloned. The chromosomal DNA of B. thuringiensis was isolated as described by Sambrook et al. (41). PCR was performed with Pyrobest DNA polymerase (TaKaRa Bio, Inc.) using the chromosomal DNA of B. thuringiensis as a template. Primers were designed according to the conserved region of the related proteins to clone the open reading frames (ORFs) of the cotJc and glnA genes, respectively. The primers were as follows: cotJc-F NdeI (5′ GGG CAT ATG TGG ATT TAT GAA AAA AA3′) and cotJc-R HindIII (5′ GGG AAG CTT AAA ATA TAT TTT TCT ATC GC3′), amplifying the 582-bp fragment, and glnA-F NdeI (5′ CGC CAT ATG TCT AGA TAC ACA AAA GAA G3′) and glnA-F XhoI (TTT CTC GAG GTA AAG AGA CAT ATA TTG ATC 3′), amplifying the 1,347-bp fragment. The cotJc and glnA gene fragments were analyzed by 1% agarose gel electrophoresis, purified, and cloned into the pET30a vector according to the manufacturer's instructions. The resultant plasmid was sequenced completely (Invitrogen, Shanghai, China). Escherichia coli transformation was carried out according to the method of Sambrook et al. (41). The heterologous expression of the cotJc and glnA genes in E. coli BL21 was performed according to the method of Yin et al. (50). The induced CotJc protein and glutamine synthetase fused with a His6 tag were purified with an ÄKTA purifier 100 system (GE Healthcare) according to the protocol of a HisTrap FF crude 1-ml column (GE Healthcare) and then used for antiserum production in rabbits as described previously (10).
Whole proteins from the three phases in B. thuringiensis 4.0718 were extracted according to the aforementioned method, except for the acetone precipitation. After protein quantification using a 2-D Quant kit, 8 mg of proteins from the three phases was separated by NuPAGE 12% Bis-Tris gel (Invitrogen) and then blotted onto polyvinylidene difluoride (PVDF) membranes (Sigma) using a tank blot apparatus (Toyo, Tokyo, Japan). The transfer, blocking, hybridization, and washing steps were performed according to standard methods (41). The PVDF membranes were incubated with primary rabbit anti-CotJc and anti-glutamine synthetase antisera at a dilution of 1:1,000 and subsequently labeled with anti-rabbit secondary antibody conjugated to goat DyLight 680 (Invitrogen) at a dilution of 1:10,000. The binding of the secondary antibody was detected using the Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, NE).


Growth morphology observations.

There have been some reports on morphological observations of B. thuringiensis at specific developmental time points, especially during spore and crystal formation, using electron microscopy (6) or laser confocal imaging (49). In the present study, we refined a new observation method using phase-contrast microscopy and obtained protein extracts from different cell types combined with a growth curve. The three different physiological phases of B. thuringiensis cells studied were the middle vegetative (T1; 10 h), early sporulation (T2; 20 h), and late sporulation (T3; 32 h), which represented the log, stationary, and declining phases, respectively. The result shows homogeneous and bulky T1 cells without spores and crystals (Fig. 1A). The T2 cells had irregular spherical spores with a few crystals (Fig. 1B). The T3 cells had regular forms as well as mature spores and crystals occupying a large volume of cells with thin cell walls (Fig. 1C).
Fig 1
Fig 1 Morphology of cells from B. thuringiensis strain 4.0718 at different phases through phase-contrast microscope. (A) Middle vegetative phase, T1; (B) early sporulation phase, T2; (C) late sporulation phase, T3. Arrows 1, spores; arrows 2, crystals.

Global analysis of the B. thuringiensis 4.0718 proteome by 2D nano-LC-MS/MS.

Currently, there is no protein database publication for the highly toxic B. thuringiensis strain whose genome is already completely sequenced. Thus, we constructed an in-house database downloaded from the Uniprot Knowledgebase consisting of 116,723 protein entries from all B. thuringiensis subspecies. By this method, we first screened 1,201 (1,034), 728 (662), and 854 (851) proteins at the T1, T2, and T3 phases in two duplicate experiments. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins of T1, T2, and T3 were identified, respectively. A total of 1,480 proteins were identified from the three phases. This represents about 14% of the published B. thuringiensis serovar kurstaki strain T03a001 proteome, which is very closely related to strain 4.0718. The pairwise comparison of the identified proteins of the T1, T2, and T3 phases shows that the commonly expressed proteins of the T1-T2, T2-T3, and T1-T3 phases were 70, 122, and 111, respectively (Fig. 2A). The numbers of commonly expressed proteins across the three phases was 308, which comprised 33.55%, 43.81%, and 39.59% of the proteins in the T1, T2, and T3 phases, respectively. On the other hand, the phase-specific proteins from the T1 to T3 phases were 429, 203, and 237, which comprised 46.73%, 28.88%, and 30.46% of the total proteins in the same order.
Fig 2
Fig 2 Distribution of the identified proteins by 2D LC-MS/MS. (A) Venn diagram for identified proteins of phases T1, T2, and T3; (B) theoretical pI distribution of the total proteome; (C) the molecular mass distribution of the total proteome.

Physiochemical characteristics of the identified proteins.

The identified proteins were then classified based on different physiochemical characteristics, such as molecular mass and isoelectric point (pI). It can be seen from the result that the molecular mass (Fig. 2B) and pI (Fig. 2C) distributions were almost similar across the three phases. The molecular masses of the most identified proteins ranged from 10 kDa to 50 kDa, and more proteins with molecular masses above 100 kDa and below 10 kDa were observed than in a typical 2D PAGE gel separation. The smallest protein identified in the T1, T2, and T3 phases was phosphatidylglycerophosphatase A (molecular mass, 1.824 Da), and the largest proteins identified for the three phases were a putative uncharacterized protein (molecular mass, 564.419 Da), polyketide synthase type I (molecular masses, 377 and 611 Da), and GTP-binding protein HflX (molecular mass, 477.255 Da), respectively. The pI values of most of the identified proteins ranged from 5.0 to 6.0, which meant that most of them were acidic proteins. About 31 (3.4%), 22 (3.1%), and 24 (3.1%) proteins had pIs greater than 10 and may be not resolvable by 2D PAGE. The most acidic protein identified in the three phases was a probable DNA-directed RNA polymerase subunit delta (pI 3.90), whereas the most basic proteins were a putative uncharacterized protein (pI 12.38), 30S ribosomal protein S11 (pI 11.53), and 50S ribosomal protein L35 (pI 11.93), respectively.

Functional classification.

The identified proteins of B. thuringiensis 4.0718 from the three phases were classified according to their annotated functions in the Uniprot Knowledgebase. Their homologies/functions were classified according to the BioCyc and KEGG metabolic pathway databases. Of the characterized part, proteins involved in small molecular metabolism comprised the largest parts, representing 33.66%, 33.43%, and 35.60%, respectively, followed by proteins associated with macromolecular metabolism which comprised 20.63% and 22.24% of the proteins from the T2 and T3 phases, respectively. Finally, proteins that participate in macromolecular metabolism in the T1 phase comprised the third largest part and accounted for 21.68% (Fig. 3A).
Fig 3
Fig 3 Functional description of the total identified protein from three phases. (A) Classes of the total identified proteins; (B) subclass of identified macromolecular metabolic protein; (C) subclass of identified small molecular metabolic protein.
The small molecular and macromolecular metabolism proteins were further sorted, depending on the metabolism phases in which they were involved. For macromolecular metabolism, 47.74%, 55.86%, and 52.20% were proteins involved in protein synthesis, degradation, and modification, which comprised the largest part among the three phases. Proteins related to DNA synthesis, degradation, and modification comprised the second largest part and accounted for 25.63%, 23.45%, and 23.70% in the three phases, respectively. The orders of the following classes were slightly different among the three phases (Fig. 3B). For small molecular metabolism, proteins that take part in amino acid metabolism comprised the largest part of the three phases and accounted for 27.83%, 23.49%, and 27.08%, followed by proteins associated with central carbon metabolism, which accounted for 13.92%, 18.72%, and 16.97% in the phases T1 to T3, respectively. Unclassified proteins comprised the third largest and accounted for 13.62% and 16.61% in the T2 and T3 phases, respectively. Those proteins that played the same role made up the fourth largest part, with 13.06% in the T1 phase. The unclassified part included proteins that participated in one or more metabolic passes. The orders of the following classes were also slightly different among the three phases (Fig. 3C). The discrepancies can be attributed to two reasons. First, metabolic flow changes in cells during development result in enzyme-related changes. Second, some proteins exist with extremely low abundances such that they cannot be detected by MS, resulting in the occurrence of a different order.
Proteins with unknown functions, including those described as genes of no characterization and hypothetical or unnamed proteins, comprised a large part and of accounted for 22.44%, 19.77%, and 16.58% of the proteins in the phases T1 to T3, respectively. This result is probably caused by the lack of a special database. Cell process proteins, including chaperones as well as those related to differentiation, sporulation, and cell mobility comprised the fourth largest part and accounted for 13.51%, 17.35, and 16.07% in T1 to T3, respectively. Proteins that participated in regulation occupied only 5.77%, 6.12%, and 5.14% of the total part because of their low abundances. Environmental information-processing proteins, including membrane transport and signal transduction proteins, accounted for 2.51%, 1.99%, and 3.47%, in phases T1 to T3, respectively. Extrachromosomal proteins mainly comprising phage-related functions and protoxins occupied the smallest proportion (see Data Sets S1 to S3 in the supplemental material).

Assessment of protoxin composition.

The entomopathogenic properties of B. thuringiensis are attributed largely to parasporal crystals, which contain one or more insecticidal protoxins produced during bacterial sporulation (44). Thus, the assessment of the protoxin composition of parasporal crystals is necessary to determine the complete potential of a particular B. thuringiensis strain. In the present study, we used a shotgun method to assess the protoxin composition of B. thuringiensis parasporal crystals.
According to the SEQUEST search results, a small amount of protoxin emerged at the early sporulation phase (T2). With continuous cell development, the types and quantities of protoxins at the late sporulation phase significantly increased. Different protoxins were suggested and sorted by the total SEQUEST score based on the identified peptides. The protoxin with the highest score was validated without any additional manual inspection. To ensure the certainty of a protoxin with a low score, this protoxin must have several discriminating peptides that have not appeared in a protoxin with a high score. Following this principle, the Cry2Aa, Cry1Ac, and Cry1Aa protoxins (shown in Fig. S3 in the supplemental material) were correctly identified in the parasporal crystals of strain 4.0718, yielding protein sequence coverages of 41.86%, 20%, and 17.11%, respectively. These results agreed with a previous report that used 1D SDS-PAGE coupled with LC-MS/MS to evaluate the protoxin composition of strain 4.0718 in our laboratory (46). As supporting information, the complete list of peptides based on which the protoxins were identified in the late sporulation phase is provided in Table S1 in the supplemental material.

Expression features of bioactive factors in strain 4.0718.

Except for ICP, the extracellular factor Vip3A and intracellular factors InhA, bacillolysin, camelysin, and S-layer proteins (SLPs) were identified in this study (Table 1).
Table 1
Table 1 Expression of proteins related to spore and crystal formation in Bacillus thuringiensis strain 4.0718 from three periods
Functional classificationProtein expressed during:
Middle vegetative phase (T1)Early sporulation phase (T2)Late sporulation phase (T3)
Bioactive factors ICPICP
Sporulation proteinsSpo0ASpo0ASpo0A
 GerBC GerBC
Spore coat proteins CotJcCotJc
Small acid-soluble spore proteins SspESspE
Crystal-related proteins ORF1 
Vegetative insecticidal protein (VIP) is a soluble protein secreted during the entire vegetative phase without evolutional homology with ICP. We identified one type of VIP, Vip3A (Q93D79), at the middle of the vegetative phase in strain 4.0718, which was continuously expressed in the latter two phases. We found that InhA (C3EHX1) and a bacillolysin protein (YP034856) are both expressed at the early sporulation phase. The camelysin proteins C3CFV1 and C3HXG9, which should be expressed earlier or simultaneously with InhA, were both identified in the late sporulation phase in the present study. Except for the SLPs that may play roles in cell integrity and shape maintenance or cell adhesion, we identified an S-layer protein, EA1 (Q63FB8), which belongs to B. cereus. This protein is highly related to the insecticidal SLP from B. thuringiensis GP1 strains, with 89% identity at the primary sequence level and 100% identity by peptide matching.

Identification of proteins involved in spore and crystal formation.

Spore and crystal formation in B. thuringiensis is a gradual process regulated by many factors (2, 8). We separated and identified proteins at different phases using 2D LC-MS/MS and found a series of related crystal and spore proteins (Table 1). Their important roles were analyzed.
The sporulation process in B. thuringiensis is likely to be very similar to the one very well characterized in B. subtilis. The presence of most key B. subtilis sporulation regulatory and structural genes is well conserved (>50% amino acid identity throughout most of the sequence). These genes include the spoO genes for the initiation of sporulation and the various sigma factors required for sequential mother cell and forespore gene transcription. Other key stage II and III sporulation genes involved in forespore formation, as well as stage IV and V regulatory genes, are also present (2). Table 1 shows the expression patterns of sporulation and regulation proteins related to the spore formation at three phases. Based on the express timeliness of the spore-related proteins, we speculate that crystal formation occurred from stages II to VI. A succession of key spore coat regulatory genes required for proper assembly, such as CotE, and many coat structural proteins, such as CotJc, were identified at the sporulation phase. The presence of these spore-related proteins plays a vital role in the assembly and cross-linking of spore (outer) coats.
Many cry genes are transcribed by both the mother cell forms of the RNA polymerase containing σE or σK. Consequently, transcription begins at stage II of sporulation by the σE form of the RNA polymerase and continues into late sporulation by the σK form of the enzyme. This dual control ensures the prolonged synthesis and thus the accumulation of large amounts of protoxins (2). In the present study, σE (Q6HER0) and σK (D5TXW6) factors were identified at early and late sporulation, respectively. Combined with previous reports, we suggest that σE can activate the expression of σK and spore genes such as spoVD and spoIVA. σK expression at the late sporulation phase reveals its critical role in the transcription of the cry gene.
Small acid soluble spore proteins (SASPs) have been proven to be specific spore components that may influence resistance to unfavorable conditions (24). Genes coding for these proteins are expressed only during the late steps of sporulation, mainly in the forespore compartment under the control of the σG RNA polymerase subunit (35). The main role of SspA (alpha type) and SspB (beta type) is to combine with the forespore total DNA to promote conformational change in DNAs in aqueous solutions and provide spore formation with necessary amino acids. The third major SASP is the SspE protein (gamma type). In contrast to the alpha and beta types, this protein exhibits only little homology among bacteria. This protein has been postulated to have different physiological roles that have not yet been identified. In the present work, we identified six types of SASPs, namely, SspE (Q71RN1), SspB (C3HKE1, Q3EP35, C3ERP0, C3EXL9, and C3D0K1), SspC (C3IWQ8 and C3H9Y2), SspD (Q63E59), Ssptlp (C3ENS1), and SspP (Q639J3). Among them, Ssptlp and SspP were expressed at the early and late sporulation phases, respectively.
The reason for the production of huge amounts of insecticidal crystal proteins in B. thuringiensis is that a mass of protoxins emerging at the early sporulation phase can be aggregated into inclusion bodies with crystal structures in mother cell cytoplasms. Generally, about 106 to 2 × 106 protoxins are transformed into crystals. The intracellular oxidative environment, which is prone to disulfide bond formation, is responsible for the correct assembly of protoxins into inclusion bodies. Many accessory proteins also participate in the process of crystal formation and its overexpression. The uncharacterized 20-kDa protein in the CryB1 5′ region (open reading frame 1 [ORF1]) and uncharacterized 29.1-kDa protein in the CryB1 5′ region (ORF2) are a class of accessory proteins. The genes encoding these proteins are usually located in the ORF of the cry2 type operon. Ge et al. (21) have confirmed that the involvement of ORF2 not only can enhance Cry2Aa expression but also can aid the formation of crystals. The repetitive units of ORF2 may provide a matrix or scaffold to crystal formation, which help misfolded ICPs to refold correctly and avoid degradation. The first gene carried by the cry2Aa operon is orf1, which shares 33% homology with the p19 gene of the cry11Aa operon. Previous studies have suggested that orf1 had no obvious effect on the expression of Cry2A (44a). In the present study, ORF2 (P21733) was only expressed at late sporulation. Hence, we speculate that ORF2 has a significant impact on the spatial structure construction of crystals at the thin stage. On the other hand, ORF1 was expressed at both sporulation phases, indicating its importance in the initial formation of crystals. Except for ORF1 and ORF2, a series of chaperones that may be related to crystal folding were identified (see Data Sets S1 to S3 in the supplemental material).

Western analysis of CotJc protein and glutamine synthetase in B. thuringiensis.

Validation of the MS results by the Western blot analysis of CotJc protein (Fig. 4A) and glutamine synthetase (Fig. 4B) revealed that the expression of CotJc protein and glutamine synthetase commenced in T2 and T1, respectively. Both continued to be expressed in the subsequent stages. The result conformed to that obtained by 2D LC-MS/MS.
Fig 4
Fig 4 Western blot analysis. (A) Western blot analysis of CotJc protein from B. thuringiensis strain 4.0718. Lane 1, middle vegetative phase; lane 2, early sporulation phase; lane 3, late sporulation phase; lane 4, positive control (the purified His6-tagged CotJc). M, molecular mass marker. The arrowhead indicates the 21.7-kDa CotJc protein. (B) Western blot analysis of CotJc protein from B. thuringiensis strain 4.0718. Lane 1, middle vegetative phase; lane 2, early sporulation phase; lane 3, late sporulation phase; lane 4, positive control (the purified His6-tagged glutamine synthetase). M, molecular mass marker. The arrowhead indicates the 50-kDa glutamine synthetase.


In this article, the proteome of B. thuringiensis 4.0718 strain at three phases (middle vegetative, early sporulation, and late sporulation) has been analyzed using non-gel-based online 2D LC-MS/MS technology. As previously reported (25), the complex nature of peptide mixtures to be analyzed on LC-MS systems often exceeds their separation capabilities. This limitation, coupled with the inherent restrictions of the automated acquisition of peptides for MS/MS, requires that samples be run more than once to increase the overall peptide identification accuracy. Totals of 918, 703, and 778 proteins were identified, which are lower than expected. This result is attributed to the lack of a protein database for B. thuringiensis, which is still far from being fully developed. Another reason is that the injection mode in 2D systems using injected salt solution plugs with increasing concentrations works at far from the optimum state of SCX. The unoptimized state of this method explains the distribution of peptides of more than one fraction, which can dilute them below their detection level or suppress their ionization in a nanoelectrospray system by higher-abundance peptides in MS analysis. However, our proposed method can overcome the intrinsic deficiency of 2D electrophoresis, as evidenced by the pI and molecular mass distributions in the present experiment. Although the theoretical pI and molecular mass values of the proteins did not reflect the actual amount due to protein modifications and the quality of databases, they provide an overview of the distribution of proteome components. Database searches remain the bottleneck for many shotgun proteomics experiments, especially when the organism database, such as that of B. thuringiensis, is not yet fully developed. Genome sequencing projects are increasing, especially for B. thuringiensis subsp. kurstaki YBT-1520 (H3abc), which is highly toxic to lepidopteran insects. Thus, use of the shotgun-based technology to study the B. thuringiensis proteome will become increasingly meaningful.
Using molecular biology methods such as PCR and RNA probes has been shown to be insufficient for the prediction of protoxin expression of B. thuringiensis. Traditional protein-based methods, such as HPLC and immunoassay, are also not ideal for identification of protoxin composition because of the high sequence homology produced in a single strain (46). Although the non-gel-based shotgun method omitted the spore-crystal mixture extraction and SDS-PAGE separation steps, it still suffers from some disadvantages, as shown by the low sequence coverages of Cry1Ac and Cry1Aa in the present study. This result can be attributed on one hand to the ineffective solubilization of the parasporal crystals of strain 4.0718 by the lysis buffers used (8 M urea buffer or guanidine-HCl buffer). On the other hand, these buffers must be subsequently precipitated using organic solvents to reduce the concentration of detergents prior to trypsin digestion to prevent the detergents from reducing the enzyme activity. Nevertheless, the solubility of the protoxins cannot always be maintained before proteolytic digestion because of their redissolution in 100 mM ammonium bicarbonate (NH4HCO3) buffer. Fu et al. (20) have established a powerful method by embedding solubilized protoxins in a polyacrylamide gel block coupled with the LC-MS/MS analysis of in-gel-generated peptides for protoxin identification, which can overcome the aforementioned disadvantages. However, our shotgun-based method was also demonstrated as a straightforward tool for the rapid and accurate assessment of protoxin composition in B. thuringiensis strains.
Vegetative insecticidal protein (VIP) is a soluble protein secreted during the entire vegetative phase without evolutional homology with ICP. The pesticidal activities of VIP can reach nanogram levels, which can be considered for new generation biopesticides (54). The immune inhibitor A (InhA) metalloprotease, which has similarities to the Bacillus thermoproteolyticus thermolysin, Pseudomonas aeruginosa elastase, and protease E-15 from Serratia, can specifically cleave antibacterial proteins such as cecropins and attacins produced by the insect host. InhA reportedly has a lethal effect following intrahemocoelic injection (34), and InhA2 is essential in providing a synergistic effect to B. thuringiensis spores on the toxicity of the Cry1C protein against Gelleria mellonella after infection via the oral route (18). In the present study, we found that InhA (C3EHX1) is expressed at the early sporulation phase (Table 1), in accordance with published reports (26). However, the late expression of InhA is not consistent with the production of antibacterial peptides, which is an initial host defense reaction (30). The role of InhA in B. thuringiensis must be further studied. Camelysin expressed during the exponential growth phase has first been purified from B. cereus. Camelysin spontaneously migrates from the surface of intact bacterial cells to preformed liposomes. The disruption of the protein-encoding gene calY results in a strongly decreased cell-bound proteolytic activity on various substrates, which proves that camelysin is an important virulence factor in B. cereus because of its interactions with the host defense and blood coagulation systems and its destruction of extracellular matrix proteins (27). Recently, our group has found that camelysin can positively regulate the expression of the InhA protein (50). The camelysin proteins C3CFV1 and C3HXG9, which should be expressed earlier or simultaneously with InhA, were both identified in the late sporulation phase in the present study (Table 1). This result is probably due to the fact that the amount of camelysin as a regulator is so low that it was not identified by MS analysis. We speculate that its apparent expression in later sporulation is related to crystal formation. Bacillolysin is a thermolysin-like exocellular zinc metal endopeptidase first isolated from Bacillus. It can catalyze the hydrolysis of the amino group peptide bond of leucine or phenylalanine. Thermolysin-like metalloproteinases, such as aureolysin, pseudolysin, and bacillolysin, have been reported as virulence factors of diverse bacterial pathogens. These specific metalloproteases are potent elicitors of innate immune responses and play important roles in killing infected hosts (1). An extracellular protease secreted by B. anthracis shares a 97.7% similarity with bacillolysin; hence, it can be classified as a bacillolysin protein. This protein is an important extracellular B. anthracis antigen factor, which can cause host bleeding and tissue destruction (12). In the present study, we identified a bacillolysin protein (YP034856) that was expressed at the early sporulation phase (Table 1), in line with the InhA expression. Chung et al. (12) have proposed that the thermolysin-like metalloproteases Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology via directly degrading host tissues, increasing barrier permeability, and/or modulating host defenses. We propose that bacillolysin and InhA may have the same effect as B. thuringiensis. The S-layer, which constitutes up to 15% of the total bacterial cell, is an ordered structure of proteinaceous paracrystalline arrays that completely cover the surfaces of many bacteria. S-layer proteins (SLPs) from Bacillus species have molecular masses ranging from 66 kDa to 255 kDa (43). Cell adhesion is the major role of SLPs. An S-layer protein coming from B. thuringiensis GP1 strains has also been reported to have high insecticidal activity against the coleopteran pest Epilachna varivestis (38). In the present article, except for the SLPs that may play roles in cell integrity and shape maintenance or cell adhesion, we identified an S-layer protein, EA1 (Q63FB8), which belongs to B. cereus. Given the expression point at the middle vegetative phase consistent with above-mentioned study, we speculate that the S-layer protein EA1 in B. thuringiensis 4.0718 has some insecticidal activity against E. varivestis. This information can be important in future insect control. However, the true role and mechanism of action of this SLP are unknown, and future work is needed to elucidate it. The aforementioned analysis revealed that the ICPs were highly expressed at the T2 and T3 phases, parallel with the expression of other bioactive factors, enabling substantial increases in B. thuringiensis toxicity. We suggest that the bioactive factor that seems to have negligible killing effects on insects may play an important role in the expression or the virulence of the ICP. The specific roles of these bioactive factors in B. thuringiensis should be further studied.
CotJc proteins are originally found in the coats of B. subtilis spores and can be encoded by the cotJ operon. The onset of cotJ transcription during sporulation coincides temporally with the appearance of the active form of σE in B. subtilis (29). Table 1 shows that some genes whose products are phenotypically implicated in coat formation, such as CotE (53) and SpoIVA (45), which occurs under the control of the early mother-cell-specific sigma factor σE, appeared together with the cotJ operon product CotJc protein at the early sporulation phase. Consequently, we speculate that the products of the cotJ operon may play a role at the early stage of coat assembly in B. thuringiensis. In B. subtilis, cotJ is not maximally induced by σE until the cellular levels of the SpoIIID regulator reach a certain threshold point. As shown in Table 1, SpoIIID is only expressed at the early sporulation phase. Thus, we propose that the SpoIIID protein is required for cotJ regulation and is not necessary for σK transcription. In the later sporulation phase, we identified five cot genes encoding coat proteins. We conclude that the bulk of the expression of the cot structural genes is dependent on σK, with the exception of cotE and cotJ. The SpoIVA protein is believed to form a basal layer around the outer surface of the forespore, which is required for the formation at the intermediate stages of the sporulation of a CotE ring that surrounds the forespore from a small distance (15). The ring of CotE is presumably held in place by a scaffold, which becomes the site of assembly of the inner coat, whereas the assembly of the outer coat takes place outside the CotE structure (15). There are no reports on cot structural genes being components of the scaffold. An interesting possibility is that cotJ products may act as components of the scaffold because the lack of an obvious phenotype of cotJ null mutants indicates only a minor function or redundancy of its encoded products (29). The role of cotJ products in B. thuringiensis must be further investigated.
The parasporal crystals in B. thuringiensis are mainly composed of 18 amino acids. Thus, nitrogen nutrition plays an important role in parasporal crystal formation and has the most far-reaching regulation effects. γ-Aminopropyl-butyric acid (GABA) metabolic pathways are mainly involved in nitrogen and carbon metabolism (19). A study has reported that the spore germination of some Bacilli is somewhat related to GABA (3). Hence, this metabolic pathway, which is accomplished via the GABA shunt, is considered to be most likely involved in the formation of spores and crystals in B. thuringiensis. Combined with the MS and Western blot results, we found that glutamine synthetase, which catalyzes the conversion of ammonia and glutamate to glutamine, commences at the early sporulation phase (Fig. 4B; see Data Set S1 to S3 in the supplemental material). As for part of the GABA shunt, we propose that the conversion between glutamate and glutamine is very important in the nitrogen nutrition metabolism of B. thuringiensis, which can further influence spore and crystal formation. Apart from glutamine synthetase, we also identified two of the most pivotal enzymes in the GABA shunt, namely, γ-aminobutyrate aminotransferase (D5TMC5) and succinate semialdehyde dehydrogenase (C3CW93), by the 2D LC-MS/MS technique. They were expressed at the early sporulation phase and continued to be expressed at the late sporulation phase (see Data Sets S2 to S3). However, we did not find glutamate decarboxylase, which can convert amino acids to GABA in vivo. This finding can be ascribed to two phenomena. First, the abundance of glutamate decarboxylase was extremely low, such that the MS analysis did not detect it sufficiently, despite the fact that the genome sequence of B. thuringiensis serovar kurstaki strain T03a001 showed that the gene coding for glutamate decarboxylase actually exists. Second, B. thuringiensis cannot synthesize GABA by itself and only uses exogenous GABA as a nitrogen source. Further studies should be carried out to prove these explanations.
The identification of the total proteome of B. thuringiensis provides only a starting point for functional analysis. Further work that combines 2D LC-MS/MS with quantitative methods, such as isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), to probe the dynamics of protein expression in B. thuringiensis is ongoing. Meanwhile, studies at the gene and metabolic levels have been carried out in our laboratory to further understand the spore and crystal biosynthesis pathway and to improve the production of the crystals.


This investigation was supported by the National Natural Science Foundation of China (30970066, 31070006, and 30900037), the National Key Basic Research and Development project (973) of China (2012CB722300), and the National High Technology Research and Development project (863) of China (2011AA10A203).

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 78Number 151 August 2012
Pages: 5270 - 5279
PubMed: 22636013


Received: 12 February 2012
Accepted: 10 May 2012
Published online: 18 July 2012


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Shaoya Huang
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Xuezhi Ding
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Yunjun Sun
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Qi Yang
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Xiuqing Xiao
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Zhenping Cao
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China
Liqiu Xia
College of Life Science, Hunan Normal University, Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, Changsha, China


Address correspondence to Xuezhi Ding, [email protected], or Liqiu Xia, [email protected].

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