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Applied and Environmental Microbiology, November 2008, p. 6811-6813, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01085-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry{triangledown} ,{dagger}

Yunjun Sun,{ddagger} Zujiao Fu,{ddagger} Xuezhi Ding, and Liqiu Xia*

College of Life Science, Hunan Normal University, Changsha 410081, People's Republic of China

Received 15 May 2008/ Accepted 3 September 2008


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ABSTRACT
 
By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.


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INTRODUCTION
 
The entomopathogenic properties of Bacillus thuringiensis are attributed largely to the parasporal crystals, which contain one or more insecticidal protoxins produced during sporulation (12). Currently, there is considerable interest in searching for novel B. thuringiensis strains with new insecticidal properties. B. thuringiensis strains typically harbor one or more cry genes, some of which are known to be cryptic. Thus, it is necessary to identify the cry gene content and the expressed protoxins concurrently in order to evaluate the potential of a particular B. thuringiensis isolate. Previous reports indicated that the combination of PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is insufficient to identify which cry genes are expressed in a strain producing multiple protoxins with similar molecular masses (8, 10). Recent examples from the literature showed that mass spectrometry (MS) can accurately analyze the protoxin compositions of B. thuringiensis strains (7, 11). In this study, we utilized a complementary method, the combination of PCR and MS, to evaluate the wild-type B. thuringiensis strain 4.0718 at both the gene and protein levels.

The wild-type B. thuringiensis strain 4.0718 (CCTCC no. M200016) was isolated from a soil sample in China (5). B. thuringiensis subsp. kurstaki strain HD1 (obtained from DSMZ, Germany) was used as the reference strain. The determination of the cry gene content of B. thuringiensis strain 4.0718 was performed by PCR with the total genomic DNA as the template. General primers designed to identify some of the main groups of cry genes were used, as follows: cry1, primers Un1(d) and Un1(r) (1); cry1I, primers S5uni and S3uni (13); cry2, primers Un2(d) and Un2(r) (1); cry3, primers CJIII20 and CJIII21 (3); cry4, primers Un4(d) and Un4(r) (1); cry5, primers gral-nem(d) and gral-nem(r) (2); cry6, primers S5un6 and S3un6 (15); cry7, primers S5un7 and S3un7 (15); cry8, primers S5un8 and S3un8 (15); cry9, primers K5un2 and S3un9 (6, 15); cry10, primers cry10A5 and cry10A3 (4); and cry11, primers gral-cry11(d) and gral-cry11(r) (2). Additionally, PCR-restriction fragment length polymorphism analysis was used to identify the subtypes of cry1 and cry2 genes as described in previous reports (6, 14).

The PCR results showed that the expected fragments of 277, 701, and 1,584 bp corresponding to the cry1, cry2, and cry1I genes were obtained in tests with 12 pairs of general primers (Fig. 1). Additionally, two PCR-restriction fragment length polymorphism results confirmed the presence of cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Thus, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1I) were detected in the strain 4.0718. The amplification and sequencing of the full-length genes showed that the sequences of the five cry genes were identical to the published sequences of cry1Aa11, cry1Ac5, cry2Aa10, cry2Ab2, and cry1Ia10 (GenBank accession no. Y09663, M73248, AF433645, X55416, and AY262167).


Figure 1
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  		FIG. 1. Agarose (1.5%) gel electrophoresis analysis of PCR products amplified from the genomic DNA of B. thuringiensis strain 4.0718 by using general primers. Lanes: M, molecular marker; 1, cry1 gene; 2, cry2 gene; and 3, cry1I gene.

The B. thuringiensis strain was grown in G-Tris medium at 30°C with shaking until cells lysed. The crystal-spore mixture of the B. thuringiensis strain was collected and treated as described previously (11). To directly determine which cry genes were expressed and crystallized in the parasporal crystals of strain 4.0718, the crystal-spore mixture was solubilized by loading buffer and a sample of about 5 µg of solubilized proteins was separated by SDS-10% PAGE. The 130- and 65-kDa protein bands on the SDS-PAGE gel (Fig. 2) were excised, in-gel digested (11), and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (9). In brief, the digested peptides were injected into an LC system (DIONEX) and first desalted and preconcentrated on a C18 PepMap precolumn (35 mm by 500 µm). The peptides were then eluted onto a C18 PepMap column (15 cm by 180 µm) coupled to the electrospray ion source of an Esquire HCT ion trap mass spectrometer (Bruker Daltonics, Germany), which is characterized mainly by its certainty in protein identification through enhanced sequence coverage. The flow rates were 20 µl/min for preconcentration and 3 µl/min for separation. The elution gradient varied from 5 to 85% buffer B (0.1% formic acid in acetonitrile) in 30 min; buffer A was 0.1% formic acid. Mass data acquisition was performed for the mass range of m/z 300 to 1,600 by using the standard enhanced mode (m/z 8,100 per s). The five most intense doubly or triply charged ions detected in each mass scan were selected and fragmented in the trap, and the resulting fragments were analyzed for mass by using the ultrascan mode (m/z 100 to 2,200 at m/z 26,000 per s). MS and MS/MS data were acquired and processed automatically for subsequent protein identification by comparison against entries in the nonredundant NCBI database for gram-positive bacteria with the use of Mascot software (www.matrixscience.com). The mass tolerances of precursor and fragment ions were set at 1.5 and 0.6 Da, and the carbamidomethylation of cysteines and methionine oxidation were set as the fixed and variable modifications, respectively. The protein identification result was considered to be significant when ions with scores higher than 41 were detected in the mass spectrum (P < 0.05).


Figure 2
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  		FIG. 2. SDS-PAGE analysis of crystal-spore mixture from sporulated B. thuringiensis strains. The gel was stained with Coomassie brilliant blue R250, and each sample was prepared in parallel. Lanes: M, molecular marker; 1, strain HD1; and 2, strain 4.0718.

The Mascot search results suggested some different protoxins, which were sorted by the total Mascot score based on the identified peptides in each band. The protoxin with the highest score was validated without any additional manual inspection. To ensure the certainty of a protoxin with a lower score, this protoxin should have several discriminating peptides which have not appeared in the protoxins with higher scores. In light of this principle, Cry1Aa and Cry1Ac protoxins were identified in the 130-kDa protein band and the Cry2Aa protoxin was identified in the 65-kDa protein band, giving protein sequence coverage levels of 49, 57, and 63%, respectively (Table 1). It was therefore confirmed that the cry1Aa, cry1Ac, and cry2Aa genes were expressed and crystallized in the parasporal crystals of strain 4.0718. As supporting information, the complete list of the peptides on the basis of which protoxins were identified is provided in Table S1 in the supplemental material.


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  		TABLE 1. Summary of results from the protoxin profiling of B. thuringiensis strain 4.0718 by SDS-PAGE coupled to LC-MS/MS

Our limited model study indicated that a combination of PCR and MS could be applied for assessing the potentials of B. thuringiensis isolates of interest. When the cry gene content in the B. thuringiensis strain was known, MS could directly determine which cry genes were expressed and crystallized in the parasporal crystal. On the other hand, for a novel B. thuringiensis strain in which PCR was unable to detect the cognate cry genes, MS could analyze the expressed protoxins so that the tryptic peptide sequence data could be used to design PCR primers specific for the novel genes.


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ACKNOWLEDGMENTS
 
This investigation was supported by grants provided through the 863 program (2006AA02Z187 and 2006AA10A212), an SRFDP grant (20060542006), and NSFC grants (30670052 and 30870064) from China.


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FOOTNOTES
 
* Corresponding author. Mailing address: College of Life Science, Hunan Normal University, Changsha 410081, China. Phone: 86-731-8872298. Fax: 86-731-8872260. E-mail: xialq{at}hunnu.edu.cn Back

{triangledown} Published ahead of print on 12 September 2008. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{ddagger} These authors contributed equally to this work. Back


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Applied and Environmental Microbiology, November 2008, p. 6811-6813, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01085-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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