Morphological Study Of Bacillus Megaterium BM2 Based On Genomic And Transcriptome Analysis

Bacillus megaterium is an aerobic gram-positive bacterium belonging to the genus Bacillus.Its shape is rod-shaped,end round,often a single or into a chain arrangement.The body is slender and relatively large compared with other microorganisms.The colony shape is regular and round,with a smooth and unwrinkled surface and a convex surface.Bacillus megaterium has been widely used in industrial and academic research fields,including microbial fertilizer,biological control,animal husbandry,water purification and expression system research and application.In the application field of Bacillus megaterium,low fermentation biomass and high production cost have always been the bottlenecks restricting its large-scale application.In the early stage of the research group,a strain of Bacillus megaterium BM2 with a small morphology and a slightly increased fermentation density was obtained in the black liquor pool.Compared with other standard Bacillus megaterium fermentations,it was found that the size of the cells and the size of the spores produced were smaller than the standard strains.Therefore,we suspect that the higher fermentation density is related to the morphology of the cells.The advantages of this strain make it a great potential for application in heterologous expression and microbial agents.This paper studies morphological and sporulation.First,genome-wide sequencing was performed to identify possible morphologically related genes in BM2 strain by genomic and comparative genomic analysis.These include the unique genes related to wall phosphonic acid synthesis(1751,1752)and genes involved in the synthesis of lipoteichoic acid(ltasa,ltasb,ltasB)and the rod-like structural protein MreB.The temperature sensitive knockout vector pKVM1 was used to construct the knockout vector,and the knockout system based on the principle of E.coli S17-1 conjugation transfer and insertion inactivation was determined by exploring the transformation methods including electrical transformation and conjugation transfer.Subsequently,gene knockout was performed on the selected morphology-related genes 1751,1752,ltasa,ltasb,ltasB and mreB.A series of morphologically related single knockout mutants(B.megaterium BM2/?1751,B.megaterium BM2/?1752,B.megaterium BM2/?ltasa,B.megaterium BM2/?ltasb,B.megaterium BM2/?ltasB,B.megaterium BM2/?mreB)and multiple knockout mutants(B.megaterium BM2/?1751?1752,B.megaterium BM2/?ltasBAltasb)were constructed.The physiological changes were observed by growth verification and the morphological changes were observed by scanning electron microscopy and transmission electron microscopy to determine the effects of these genes on cell growth,cell wall formation and cell morphology.Through physiological,biochemical and morphological analysis of each knockout strain,we found that single or multiple deletions of genes 1751,1752,ltasa,ltasb,ltasB caused slower growth of the cells.However,the deletion of the mreB gene increases the biomass of the strain in the fermentation medium.In terms of cell morphology,it can be seen by scanning electron microscopy that the deletion of the above genes affects the size and shape of the cells to varying degrees.For example,the deletion of genes related to the synthesis of teichoic acid causes the cells in the logarithmic growth phase to become larger(Itas)or become small(1751,1752),the deletion of the mreB gene causes severe inhibition of spore formation,and the cell morphology appears square or spherical.In addition,both TEM and Western Blot experiments indicate that the loss of the above genes causes changes in cell wall composition,which is one of the possible causes of growth and morphological changes.These findings indicate that the morphology-related genes we preliminarily screened have certain effects on the growth and cell size of bacteria,and some genes are related to the formation of spores.Finally,we analyzed the changes in gene expression levels of B.megaterium BM2 during the growth cycle by transcriptome.On the one hand,the difference in the expression level of the morphologically related genes screened in this paper was verified at the level of gene expression.On the other hand,it provides data support for further exploration of other genes involved in cell morphology and sporulation.