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Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

Rang J, He H, Wang T, Ding X, Zuo M, Quan M, Sun Y, Yu Z, Hu S, Xia L - PLoS ONE (2015)

Bottom Line: The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry.For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons.A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

No MeSH data available.


Related in: MedlinePlus

Protein identified of phases T1, T2 and T3 by mass spectrometry compared with the results of genome annotation.The part of circle outer represents the proteins that can be detected by LC-MS/MS but can not be searched from the results of the genome annotation.
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pone.0119065.g001: Protein identified of phases T1, T2 and T3 by mass spectrometry compared with the results of genome annotation.The part of circle outer represents the proteins that can be detected by LC-MS/MS but can not be searched from the results of the genome annotation.

Mentions: B. thuringiensis 4.0718 protein expression in proteomics was analyzed. Shaoya Huang has already performed this proteomics experiment in our laboratory, and thus, the analysis in this section mainly refers to the related proteomics analysis obtained from his study. The results from his study were compared with our genome annotation results. In Shaoya Huang’s research, 1,201 (1,034), 728 (662), and 854 (851) proteins at the T1 (10 h), T2 (20 h), and T3 (32 h) phases were screened, respectively, in two duplicate experiments. After deleting redundant proteins from different batches of B. thuringiensis subspecies, 918, 703, and 778 proteins in the T1, T2, and T3 phases were identified, respectively [15]. A total of 1,480 proteins were identified from the three phases, which accounted for 21.94% of the total protein post-genome annotation. Protein expression quality percentages at the three respective stages were 13.57%, 10.39%, and 11.50% of the encoded sequence of the B. thuringiensis 4.0718 genome. Upon comparison analysis, only three pesticidal Cry proteins (Cry2Aa, Cry1Aa, and Cry1Ac) were detected in his study. However, the genome annotation result shows that B. thuringiensis 4.0718 also contained other pesticidal Cry proteins, such as Cry2Ab and Cry1Ia (Table 3). By analyzing the positions of these five Cry protein genes in the genome, these genes were found to be located in two plasmids, and no Cry protein genes have been detected in the chromosome genome. Comparing the LC-MS/MS results to match the genome annotation results, few proteins were found each time we were unable to find or match the LC-MS/MS with the genome annotation results (Fig. 1, Tables 4 and 5). Generally, the unmatched proteins were mainly at different locations of the initiator methionine (Fig. 2). Therefore, the predicted result altered the number of methionine. Methionine also served an important function in the formation of either intra- or inter-chain disulfide bonds. Eventually, the analysis of the spatial structure of the protein will reveal differences.


Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718.

Rang J, He H, Wang T, Ding X, Zuo M, Quan M, Sun Y, Yu Z, Hu S, Xia L - PLoS ONE (2015)

Protein identified of phases T1, T2 and T3 by mass spectrometry compared with the results of genome annotation.The part of circle outer represents the proteins that can be detected by LC-MS/MS but can not be searched from the results of the genome annotation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363619&req=5

pone.0119065.g001: Protein identified of phases T1, T2 and T3 by mass spectrometry compared with the results of genome annotation.The part of circle outer represents the proteins that can be detected by LC-MS/MS but can not be searched from the results of the genome annotation.
Mentions: B. thuringiensis 4.0718 protein expression in proteomics was analyzed. Shaoya Huang has already performed this proteomics experiment in our laboratory, and thus, the analysis in this section mainly refers to the related proteomics analysis obtained from his study. The results from his study were compared with our genome annotation results. In Shaoya Huang’s research, 1,201 (1,034), 728 (662), and 854 (851) proteins at the T1 (10 h), T2 (20 h), and T3 (32 h) phases were screened, respectively, in two duplicate experiments. After deleting redundant proteins from different batches of B. thuringiensis subspecies, 918, 703, and 778 proteins in the T1, T2, and T3 phases were identified, respectively [15]. A total of 1,480 proteins were identified from the three phases, which accounted for 21.94% of the total protein post-genome annotation. Protein expression quality percentages at the three respective stages were 13.57%, 10.39%, and 11.50% of the encoded sequence of the B. thuringiensis 4.0718 genome. Upon comparison analysis, only three pesticidal Cry proteins (Cry2Aa, Cry1Aa, and Cry1Ac) were detected in his study. However, the genome annotation result shows that B. thuringiensis 4.0718 also contained other pesticidal Cry proteins, such as Cry2Ab and Cry1Ia (Table 3). By analyzing the positions of these five Cry protein genes in the genome, these genes were found to be located in two plasmids, and no Cry protein genes have been detected in the chromosome genome. Comparing the LC-MS/MS results to match the genome annotation results, few proteins were found each time we were unable to find or match the LC-MS/MS with the genome annotation results (Fig. 1, Tables 4 and 5). Generally, the unmatched proteins were mainly at different locations of the initiator methionine (Fig. 2). Therefore, the predicted result altered the number of methionine. Methionine also served an important function in the formation of either intra- or inter-chain disulfide bonds. Eventually, the analysis of the spatial structure of the protein will reveal differences.

Bottom Line: The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry.For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons.A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

No MeSH data available.


Related in: MedlinePlus