Proteomic analysis of Bacillus thuringiensis at different growth phases by using an automated online two-dimensional liquid chromatography-tandem mass spectrometry strategy

Abstract

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.

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.

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.

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.

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.