Effects Of Two Enzyme Genes On Biofilm Associated Phenotypes Of Bacillus Thuringiensis

Bacillus thuringiensis(Bt)formulations have been applying as one of the most widely used microbial pesticides on the market.However,its bacterial cells and the active ingredients are relative easily inactivated under sunlight(ultraviolet rays,UV),which limits its field duration.Bacterial biofilm(BBF)is a group of cells grown on solid-liquid,solid-air,liquid-liqud or liquid-air interfaces and enclosed in an extracellular polysaccharide matrix,which can increase the resistance of bacteria to environmental stresses such as UV.Therefore,investigation of Btbiofilms may open up new solutions for the above bottleneck.Using comparative genomics,BtXL6 genes that may be involved in the formation of BBF were discovered in our previous work.In this study,the effects of two such genes,BTXL6＿18630 and BTXL6＿25120,on the biofilm associated phenotypes of Btwere investigated.The phenotypic change in biofilm formation,UV resistance,motility,growth rate and spore germination provided a theoretical basis for the subsequent researches related to the elucidation of regulatory network of Btbiofilms and construction of Btbiofilm engineering strains with high UV-resistance.The main results of this study are as the follows:The functions of BTXL6＿18630 gene and BTXL6＿25120 gene were analyzed by bioinformatics.The results showed that the open reading frame(ORF)of BTXL6＿18630 was 795 bp in length and had 99%homology with UDP-galactose-lipid carrier transferase gene from Btand Bacillus cereus(Bc);it encoded a 31.96 k Da protein with 264 amino acids,and was a hydrophobic stable protein without any signal peptide and transmembrane structure,indicating the protein played its role in the cytoplasmic matrix;and it belonged to polyphosphate kinase 2(PPK2)superfamily,which includs polyphosphate kinase 2(PPK2)and inorganic polyphosphate(poly P).On the other hand,the ORF of BTXL6＿25120 was2406 bp in length and had 99% homology with PAS domain S-box protein in Btand Bc;it encoded a non-secreted 91.4 k Da protein with 801 amino acids,and was a hydrophilic stable protein without any signal peptide but had 5 transmembrane regions,indicating it may responsible for the change in the cell membrane.;and it was a homolog with 4 superfamilies consisting of 5TM-5TMR＿LYT,PAS,PAS domain S-box and Bae S.In order to further study the effects of single gene deletion of BTXL6＿18630 or BTXL6＿25120 on the biofilm associated phenotypes of BtXL6,overlapping PCR technology was used to construct gene knockout vectors p MAD△XL6＿18630ΩKm and p MAD XL6＿25120ΩKm.The two vectors were introduced into Btwild type strain XL6 using electrotransformation technology.Gene knockout were induced by homologous recombination at 46°C to obtain mutants BtXL6 △18630ΩKm and BtXL6△25120ΩKm.In addition,promoters and ORFs of BTXL6＿18630 and BTXL6＿25120 were amplified by PCR to construct gene complementary vectors p HT18630 and p HT25120,respectively.Subsequently,BtXL6 △ 18630ΩKm and BtXL6 △ 25120ΩKm were transformed by electrotransformation to obtain the gene complementary strains BtXL6△18630ΩKm::18630 and BtXL6△25120ΩKm::25120,respectively.The phenotypic change in BBF formation,UV resistance,motility,growth rate,and spore germination of BtXL6 caused by gene deletion of BTXL6＿18630 or BTXL6＿25120 was investigated.The results showed that BTXL6＿18630 and BTXL6＿25120 genes did not affect the growth rate of BtXL6.The wild type strain BtXL6 formed a robust BBF(submerged bottom type BBF)in LB medium,while in MSgg medium a thin layer of incomplete BBF was formed on the solid-liquid interface.Using a confocal laser scanning microscope,it was found that the average thickness of BtXL6 △ 25120ΩKm biofilm(43.7955±6.3277 μm)was significantly higher than that of the wild type strain(33.1810 ± 2.6215μm)(p<0.05),whereas BTXL6＿18630 gene did not affect the biofilm thickness of BtXL6.BTXL6＿25120 negatively regulated the swimming ability of BtXL6,and positively regulated the tyrosine arylamine enzyme activity and the usage of N-acetyl-D-glucosamine.BTXL6＿18630 and BTXL6＿25120 did not affect the spore germination and UV resistance ability of the strains in planktonic or BBF conditions.In order to clarify the fundamental functions of the proteins encoded by BTXL6＿18630 and BTXL6＿25120,expression vectors p ET32a-18630 and pET28a-25120 were constructed and the proteins were expressed in Escherichia coli.SDS-PAGE results showed that p ET32a-18630 was successfully expressed a 49.7 k Da soluble protein BTXL6＿18630.However,the BTXL6＿25120 protein expressed by p ET28a-25120 had always been presented in the form of inclusion bodies.In summary,this study clarified the difference in the BBF formation ability of BtXL6 in different media,and elucidated the impacts of the single gene deletion of BTXL6＿18630 and BTXL6＿25120 on the BtXL6 biofilm associated phenotypes,providing a theoretical basis for our understanding of biofilm regulation networks and the construction of Btbiofilm engineering strains with high resistance to UV.