Comparative analysis of genomics and proteomics in Bacillus thuringiensis 4.0718

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.

Fig 1. 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.

Fig 2. Analysis of start codon and initiator methionine for acetate CoA-transferase, alpha subunit.

Red arrow, initiator methionine, as identified by Prodigal.v2_60 software. Green arrow, initiator methionine of the protein identified by LC-MS/MS through received uniprot blast. These initiator methionines were determined from B. thuringiensis subsp. konkukian (strain 97–27) and B. thuringiensis serovar kurstaki strain T03a001, respectively.

Fig 3. Complete circuit diagram of key regulatory genes in sporulation.

Spo0A is a key protein that directs the transcriptional regulation of downstream gene, including asymmetric division and the expression of SigF and SigE, which are special transcription factors of pre-spore and mother cell, respectively. The two transcription factors decide the regulation of the development of spore. Red line, inhibition; Orange line, activation; Green, translation.

Fig 4. Analysis of insecticidal Cry protein regulation network.

The figure exhibits that the insecticidal Cry protein interacts with beta-lactamase type II. Beta-lactamase type II is also regulated by LysR family transcriptional regulator.