Functional Study Of C-terminal Signal Peptidase CHU₃237 And Chu_gld1,chu_gld2,chu_ion1 In Cytophaga Hutchinsonii

Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently and rapidly degrade cellulose,but its unique mechanism of cellulose degpradation remains an unsolved mystery.In recent years,with the continuous research on C.hutchinsonii,the functions of many genes affecting cellulose degradation have gradually become clear,and its unique cellulose degradation mechanism is being gradually clarified.Therefore,further research on C.hutchinsonii's cellulose-related genes will help reveal the cellulose degradation mechanism of C.hutchinsonii and promote cellulose utilization.The protein CHU₃237 of C.hutchinsonii which has a Peptidase family C25 domain and is predicted to be a homologous protein to the C-terminal signal peptidase PorU in P.gingivalis T9SS.Previous research in the laboratory showed that after the gene chu₃237 was knocked out,the mutant ?3237 had partial deletions in cellulose utilization and sliding ability,and a significant reduction in extracellular proteins,many of which were predicted to be substrates of the T9SS.Therefore,it is predicted that CHU₃237 also works as a peptidase that cleaves CTD in C.hutchinsonii.In order to further verify the function of CHU₃237,this paper mutated the functional site of its possible protease,and studied various properties of the mutant strain.In addition,the function of other genes that may be involved in cellulose degradation was also studied.Genes chu_gldl,chu_gld2,and chu_ion1,which may affect cellulose degradation,were screened by transposon mutations in the early stage.In this paper,the genes were knocked out by homologous double-exchange method to study the effect of the deletion strain on cellulose degradation.The specific research contents and results are as follows:1.Functional study of CHU₃237By consulting the literature and bioinformatics analysis,it is inferred that the active site of CHU 3237 is histidine 803 and cysteine 837.Two in-situ single and double mutations of CHU₃237 protein are obtained,Mutants H803A,C837A,H803A&C837A.It was found that the mutants H803A,C837A,H803A&C837A had a significant decrease in the filter paper degradation ability and the agar plate diffusion ability,and the cell surface enzyme activity also significantly decreased,while the extracellular protein secretion was significantly reduced,of which the major extracellular secreted protein CHU₀344 significantly reduced the extracellular protein content.The localization of cell surface cellulase CHU₀778 and CHU₁336 secreted by T9SS was further studied.The results of westemblot showed that protein modification may exist in CHU₀778 and CHU 1336 during the secretion process,while the deletion of CHU 3237 affected the maturation of positioning.These results indicate that the absence of CHU₃237 affects the maturation and localization of C.hutchinsonii cell surface proteins and extracellular protein secretion,which in turn affects the cellulose degradation and slip of C.hutchinsonii.We further examined the transcription and expression of CHU 3237 in the mutant strain,and the results showed that point-mutated CHU₃237 could perform normal transcription and expression.These results indicate that amino acids 803 and 837 of CHU₃237 are necessary for the function of CHU₃237,and CHU₃237 may have protease activity.We added the His_Tag tag to heterologously express CHU 3237 in E.coli and tried to test the enzyme activity in vitro.However,due to the very limited protein expression,we didn't detected activity in vitro.2.Preliminary study on function of chu_gld1,chu_gld2,chu_ionlThe genes chu_gld1,chu_gld2,and chu_ion1 that may be involved in cellulose degradation were knocked out one by one.After the deletion of chu_gld1,chu_gld2,and chu_ion1,the use of cellulose by the mutants was delayed to varying degrees,indicating that the deletion of chu_gld1,chu_gld2,and chu_ion1 caused C.hutchinsonii's ability to degrade cellulose decreased,but it did not completely lose its ability to degrade cellulose.Further research showed that the mutations in each gene did not directly affect the cellulase activity of cells and their ability to adsorb crystalline cellulose,and the mechanism of its impact on cellulose degradation needs to be further explored.The effect of chu_gldl and chu_gld2 on cellulose utilization may be related to its effect on cell movement.Further research on the mechanism of chu_gldl and chu_gld2 affecting cell movement will help reveal the relationship between C.hutchinsonii sliding ability and cellulose degradation.