The metabolic regulation of sporulation and parasporal crystal formation in Bacillus thuringiensis revealed by transcriptomics and proteomics

Abstract

Bacillus thuringiensis is a well-known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20-30% of the cell's dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: (1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; (2) stored poly-β-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; (3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; (4) the tricarboxylic acid cycle was significantly modified during sporulation; (5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; (6) most F0F1-ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels.

Fig. 1.

Growth features of strain CT-43. A, The growth curve of strain CT-43 grown in GYS. The y-axis presents the average optical densities of triplicate bacterial cultures at 600 nm at each time point. Data are averages of at least three independent experiments (error bars are S.E. from mean values).The four sampling points of 7 h, 9 h, 13 h, and 22 h for transcriptomics and proteomics are scaled out. B, Images of cell growth status at four time points of 7 h, 9 h, 13 h, and 22 h. The spore and parasporal crystal are marked. The scale bars represent 10 micrometers.

Fig. 2.

COG analysis. A, Functional classification of the genes expressed at each time point in RNA-seq data. The “7 h, ” “9 h, ” “13 h, ” and “22 h ” indicate the numbers of the transcribed genes at each growth phase, whereas the “total ” represents the number of the total genes encoded by the CT-43 chromosome. B, Functional classification of the quantified proteins in each temporal comparison in iTRAQ data. The “7 h versus 9 h”, “7 h versus 13 h”, “7 h versus 22 h”, “9 h versus 13 h”, “9 h versus 22 h”, and “13 h versus 22 h ” represent the numbers of the quantified proteins in each temporal comparison. COG designations are described as follows: A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell division and chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme metabolism; I, Lipid metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, DNA replication, recombination, and repair; M, Cell envelope biogenesis, outer membrane; N, Cell motility and secretion; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolite biosynthesis, transport, and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking and secretion; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton; None, No COG information.

Fig. 3.

The contents of 20 common amino acids in strain CT-43. A, The contents of 20 common amino acids in a total of 6626 proteins encoded by one chromosome and 10 plasmids of CT-43. B, The contents of 20 common amino acids in five ICPs of CT-43. The number above each column represents the percentage of corresponding amino acid. The branched-chain amino acids (BCAAs), including isoleucine, leucine and valine, have very high percentages in both total 6626 proteins and five ICPs.

Fig. 4.

Biosynthesis and reuse of PHB in CT-43. A, The transcriptional level of the main genes associated with the synthesis and degradation of PHB. B, changes in the PHB level. C, PHB granules during different phases. Under a phase contrast microscope, CT-43 parasporal crystals are diamond- or spindle-shaped, whereas the PHB granules have an irregularly spherical shape. The level of intracellular PHB was measured as described previously (34). Data are averages of three independent experiments (error bars are S.E. from mean values). The photographs were obtained using a phase contrast microscope (Nikon ECLIPSE E6000). The PHB particles are marked. The scale bars represent 10 micrometers.

Fig. 5.

The expression features of the ilvEBHC-leuABCD operon and the leuB gene. A, The transcriptional level of the ilvEBHC-leuABCD operon at 7 h, 9 h, 13 h, and 22 h using the RPKM value of each gene as the y-axis. B, Frequencies of mapped bases of the ilvEBHC-leuABCD operon at 13 h. C, The upstream regulatory region within 400 nt upstream of the ORF leuB initiation codon ATG. The number of unambiguously mapped reads per nucleotide was calculated and visualized by R and Origin version 8.0. Each ORF is depicted with corresponding direction and length.