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. 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 ␦-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 c...

Changes in lipid and proteome composition accompany growth ofBacillus subterraneusMITOT1 under supercritical CO2and may promote acclimation to associated stresses

“…11). Observation of proteins predicted to participate in acetoin metabolism (Fig_989, Fig_1512 and Fig_3152) are consistent with their previous observation in stationary-phase (but not exponential-phase) proteomes of B. thuringiensis (55) and may reflect the role of acetoin as a secondary carbon source during stationary phase growth of multiple bacterial types (64). In addition, the MITOT1 proteomes revealed a carbon starvation protein (Fig_2079), the cell division protein, FtsH (Fig_3548) and two cold shock proteins (Fig_446 and Fig_3723) which have also previously been associated with proteomes of stationary phase cultures (65, 66).…”

“…6). These protein counts are similar to those recovered from other Bacillus proteomes (within a factor of 2) (54, 55), despite limitations due to low biomass densities obtained from anaerobic cultivation.…”

“…Indeed, observed cell density and protein expression across headspace and pressure/incubation conditions are consistent with stationary phase growth. Congruence of highly expressed proteins in scCO 2 -exposed proteomes with other Bacillus proteomes from stationary phase cultures (55, 56, 71) is notable as it suggests that B. subterraneus MITOT1 can acclimate and maintain housekeeping metabolic processes under a scCO 2 headspace.…”

Comparative Analysis of Genomics and Proteomics in Bacillus thuringiensis 4.0718

“…The process of sample preparation, extraction and digestion of whole-cell proteins, and 2D nano-LC-MS/MS (ThermoFisher, USA) analysis are using previously described methods [ 15 ].…”

“…Huang et al reported the expression of other proteins in B . thuringiensis by studying proteomics at different growth phases using liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) and carried out preliminary analysis for the associated proteins [ 15 ]. Traditionally, LC is the most widely used proteomic method for analyzing complex protein mixtures prior to MS analysis [ 16 , 17 ].…”