Functional Study Of Chu₂177,chu₂178,chu₁927,Chu₂532 And Chu₃392 Related To LPS Synthesis Of Cytophaga Hutchinsonii

Cytophaga hutchinsonii is an aerobic gram-negative bacterium that exists widely in the soil.It belongs to the phylum Bacteroidetes.It uses a new and unknown method to degrade crystalline cellulose,which is different from free cellulase system and cellosome.In addition,C.hutchinsonii can use an unclear movement mechanism to glide on the surface of solid media,which does not depend on flagella or pili.The type IX secretion system(T9SS)is widely distributed and only exists in the phylum Bacteroides.The most researched is the secretion effect of T9SS of Porphyromonas gingivalis on protein.The protein with C-terminal conserved domain(CTD),including its main virulence factor cysteine protease,is secreted by T9SS to the cell surface where they are anchored via anionic lipopolysaccharide(A-LPS).T9SS and A-LPS are critical to the pathogenicity of P.gingivalis.C.hutchinsonii has all homologous proteins of T9SS.Previous studies have found that the core components of T9SS play an important role in cellulose degradation and sliding,and many CTD proteins secreted by T9SS play a key role in cellulose degradation.However,whether C.hutchinsonii has A-LPS and the anchoring mechanism of CTD proteins secreted by T9SS are still unclear.In this study,more than 10,000 transformants were obtained by the method of transposon insertion mutation,and 54 strains deficient in filter paper degradation were selected.The insertion site was identified by inverse PCR technology.After bioinformatics analysis,32 genes were selected for single knockout,and 5 mutants were found to be related to the synthesis of LPS.The functions of these 5 genes were further studied,and the mechanism of LPS affecting cellulose degradation was explored.The main research contents are as follows:The functional study of chu₂177and chu₂178 in C.hutchinsonii.According to the annotation of the function predicted by NCBI,chu₂177 encodes the O-antigen ligase,the downstream gene chu₂176 encodes possible exporter of O-antigen and teichoic acid,and the upstream gene chu₂178 encodes the conserved hypothetical protein.There is also a chu₀602 gene predicted to encode O-antigen ligase in C.hutchinsonii.Through the analysis of bioinformatics and LPS silver staining maps of each strain,it was confirmed that chu₂176 encodes the O-antigen flipase,chu₂177 encodes the O-antigen polymerase,chu₂178 encodes the O-antigen chain length determinant protein,and chu₀602 encodes the O-antigen ligase.These genes are involved in the synthesis of LPS,and only one type of LPS is produced in C.hutchinsonii.In addition,the ?2177 and ?2178 mutants cannot degrade filter paper and crystalline cellulose,have sliding defects,significantly reduce the adsorption capacity of cellulose,reduce extracellular polysaccharides,obstacles to biofilm formation,and decline in stress resistance.The cellulases of C.hutchinsonii are mainly located in the outer membrane of the cell.The cellulase activities in the outer membrane of the ?2177 and ?2178 mutants were significantly reduced,while the extracellular cellulase activity increased.It is inferred that the cellulases failed to anchor in the outer membrane of the mutants and released to the extracellular.Outer membrane proteins and extracellular proteins of the wild type,?2177,and ?2178 mutants were extracted and identified by mass spectrometry.It was found that the outer membrane CTD proteins of ?2177 and ?2178 decreased by about 60%,and the extracellular CTD proteins increased significantly.The reduced CTD proteins are A-type CTD proteins,including CHU 0922 that affects cellulose degradation,3 endocellulases and 7 glycoside hydrolase family proteins,which speculated that A-type CTD proteins of C.hutchinsonii may be anchored to the outer membrane through LPS.Therefore,we used the endocellulase CHU₁336 antibody to locate CHU₁336,and found that CHU₁336,which is located in the outer membrane of the wild type,was not detected in the outer membrane of the ?2177 and?2178 mutants,but appeared in the extracellular matrix,proving the defect of LPS will affect the anchoring of some A-type CTD proteins in the outer membrane,including cellulase.The study also found that the apparent molecular weight of some outer membrane proteins without CTD in the mutants were smaller than that of the wild type.It is speculated that these outer membrane proteins may have LPS modification.After extracting LPS of the wild type and measuring its monosaccharide components,it was found that mannose,ribose,galacturonic acid and fucose may be present.We tested the outer membrane proteins of WT,?2177 and ?2178 with the aleuria aurantia lectin(AAL),which can recognize fucosylated proteins,and found that WT has a large amount of fucosylated proteins,while no glycoproteins was detected in the outer membranes of the ?2177 and ?2178 mutants,further indicating some outer membrane proteins without CTD may be glycosylated by LPS.The mechanism of LPS glycosylation modification of non-CTD proteins in the outer membrane of C.hutchinsonii remains to be further studied.We speculate that after the O-antigen of LPS is synthesized and flipped to the periplasmic space,in addition to participating in the synthesis of LPS,it may also be transferred to proteins by oligosaccharyltransferase to form glycosylated proteins.In summary,we found that C.hutchinsonii only has one type of LPS.The defective LPS affects the LPS glycosylation modification of the outer membrane proteins without CTD and the anchoring of the CTD proteins on the cell surface,which reduces the cellulase activity and cellulose adsorption capacity,thereby affecting the degradation of cellulose.The functional study of chu₁927,chu₂532 and chu 3392 in C.hutchinsonii:Bioinformatics analysis predicts that chu₁927 encodes the initiation enzyme for synthesizing O-antigen,chu₂532 encodes the glycosyltransferase for synthesizing O-antigen,and chu₃392 encodes the dehydrogenase for synthesizing sugar precursor of O-antigen.Knockout of chu₁927,chu₂532 and chu₃392 alone resulted in the loss of the O-antigen of LPS,indicating that chu₁927,chu₂532 and chu 3392 are involved in the synthesis of O-antigen,and further confirmed that C.hutchinsonii only produces one type of LPS.The ?1927,?2532,?3392 mutants cannot degrade filter paper and crystalline cellulose,have sliding defects,reduce the adsorption capacity of cellulose,reduce extracellular polysaccharides and obstacles to biofilm formation.In addition,it was also found that the apparent molecular weight of many outer membrane proteins in the mutant strain are smaller than that in the wild type,indicating that these proteins may have LPS modification.This article has discovered and discussed the mechanism of LPS affecting the degradation of cellulose in C.hutchinsonii,which will provide us with a powerful help to further clarify the unique cellulose degradation mechanism of C.hutchinsonii,and will also promote the efficient use of cellulose resources.