Effects Of The Synthesis And Degradation Of Poly (β-Hydroxybutyrate) On Sporulation And Insecticidal Crystal Proteins Formation In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is a ubiquitous Gram-positive,rod-shaped and sporulating bacterium which produce a wide variety of parasporalcrystalline proteins often within secticidal or nematicidal properties against larvae of very diverse insect orders.Poly-3-hydroxybutyrate(PHB)is accumulated intracellularly in the form of inclusion bodies(PHB granules)during times of over supply with carbon sources and/or in the absence of one or more essential nutritional elements.The biochemical pathway leading from central intermediates to PHB is rather simple and requires only thiolase Pha A,reductase Pha B and PHB synthase(Pha C).As for its degradation,also requires three enzymatic steps and catalyzed by PHB depolymerase(Pha Z),3-hydroxybutyryl-Co A dehydratase(Bdh A)and β-ketoacyl-Co A thiolase.PHB was accumulated maximally just prior to the formation of spores and was degraded during the process of sporulation.So many researches reported that the main role of the polymer was to serve as carbon and energy sources to fuel the sporulation process.Through the analysis of the distribution of the genes that encoding PHB metabolism related enzymes in 3500 kinds of prokaryotes,we found that there existed 760 strains have PHB synthesis and degradation related genes.Most of Bacillus species such as Bacillus subtilis are natural PHB-negative phenotype,and bioimformatic analysis reveals that no known pha genes or sequences occur in their genomes,indicating that PHB has no direct correlation with sporulation.To investigate the relationship between PHB accumulation and synthesis of ICPs and sporulation in the absence of pha C and pha Z,we constructed pha C and pha Z deletion mutants Δpha C and Δpha Z by markerless gene deletion method.Because BMB171 was an acrystalliferous strain used in the study,shuttle vector p BMB-cry1Ac10 containing the ORF of insecticidal crystal protein gene cry1Ac10 was used to transform BMB171,Δpha C and Δpha Z,the obtained transformants were named as BMB171-cry,Δpha C-cry and Δpha Z-cry,respectively.Though the preliminary testing of phenotypes of the strains,we found the growth curves of BMB171,Δpha C and Δpha Z in GYS medium had no obvious difference between each other.Cell morphology during exponential phase and sporulation phase was observed under transmission electron microscope.The cell size and shape of the mutants were in accordance with those of the parent strain.To explore the relationship between PHB accumulation and sporulation in B.thuringiensis,we counted the spore formation efficiency in LB medium and found there were no significantly different in BMB171,Δpha C and Δpha Z strains.And we also investigated the concentration of ICPs and the bioassay against cotton bollworm,which proved no significantly changes among these strains.It showed that PHB accumulation in the pha C mutant was totally abolished.However,cell morphology during exponential phase and sporulation phase was observed under transmission electron microscope.It existed some PHB granules only in a few dead cells of Δpha Z-cry and most of the strains could degraded PHB.Consequently,we speculated that some other degradation mechanism may presented in Δpha Z-cry,which lead to the death of undegradable cells and survival of degradable cells.Consequently,there is no relationship between PHB accumulation and synthesis of ICPs and sporulation in B.thuringiensis through the markerless gene deletion of pha C or pha Z.In the case of PHB deficiency,PHB biosynthetic pathway competes for acetyl-Co A with other metabolic pathways such as the tricarboxylic acid(TCA)cycle and fatty acid biosynthesis pathway that consumes acetyl-Co A,thereby leading to the reduced availability of acetyl-Co A for PHB biosynthesis.Under the condition of pha Z deletion,most of bacterial cells can still degrade and use PHB through unknown mechanisms.