| Cytophaga hutchinsonii belongs to the gram-negative aerobic bacteria categorized into the phylum Bacteroidetes.It is widely present in the soil and can rapidly degrade cellulose and slide.But different from the two currently known cellulose degradation strategies,C.hutchinsonii neither secretes free cellulase nor forms fibrosomes on the cell surface,and it does not have flagella or fimbria,the unique mechanism of degradation and slippage remains secret so far.The third degradation mechanism deems there is a protein complex on the cell surface.When the bacterial cells directly contact with cellulose,the complex can peel off the cellulose chain and transport it to the periplasmic space for further degradation.Previous studies in our lab found that C.hutchinsonii has two ways to degrade cellulose happened in cell surface and periplasmic space,and the outer membrane proteins play essential roles in cellulose binding and degradation in cell surface.Therefore,the normal modification and anchoring of outer membrane proteins on the cell surface are essential in the degradation of cellulose.T9SS（TypeⅨ of secretion system）only exists in the phylum Bacteroides.There are C-terminal domains（CTDs）in proteins secreted by T9SS.These proteins are first transported across the cytoplasmic membrane via the Sec transport system,and then translocated across the outer membrane by T9SS directed by the CTDs to the cell surface or extracellular.In C.hutchinsonii,it has been found that in addition to cellulases located in cell surface,there are many proteins with CTDs that play an important role in the adsorption and degradation of cellulose.However,how these proteins are anchored to the outer membrane is still unknown.In this work,a number of genes that affect cellulose degradation were screened by transposon insertion mutation technology,and the functions of three genes involved in lipopolysaccharide synthesis,chu2777 and chu1044 were studied.We preliminarily determined the anchoring method of protein on the cell surface of the T9SS cargo proteins.The specific research contents and the results obtained are as follows:C.hutchinsonii O-antigen flipase functions study:The gene chu2777（rfbX）was screened by insertion mutation,which may be O-antigen flipase,located in the cytoplasmic membrane and belongs to the MATElike super family.It has 12 transmembrane domains typical of this family protein.The bioinformatics analysis found that there are three other MATElike super family proteins,chu2176,CHU2507,and CHU2883,which were delected and verify their phenotypes.Delete chu2777 and chu2176 respectively,the mutant strains were unable to degrade the filter paper.Under liquid culture conditions,the ability to degrade Avicel and RAC was weakened,gliding ability was lost,O-antigen of LPS was missing,but the phenotypes above of Δ2507 and Δ2883 are the same as WT,indicating that chu2507 and chu2883 may be redundant genes,which are not involved in the synthesis process of LPS,or flip other types of polysaccharide units.The cell surface endocellulase activity of Δ2777 and Δ2176 is almost completely lost.The endocellulase activity of lysing cells decreases most obviously in the early growth stage,which is about 50%less than that of WT.β-Glucosidase enzyme activity on the cell surface and lysed cells is about 40%less than WT in the early stage.The cellulase activity electrophoresis showed that the cellulase on the cell surface of the deletion strains were significantly reduced,but on the extracellular fraction were higher than WT.Through cell surface proteomics,47 A-type CTD proteins（TIGR04183）were identified on the cell surface of WT,22 of which were missing from the cell surface of Δ2777,and 28 were missing from the surface ofΔ2176,including cellulase CHU1336 and CHU1335.Western blot experiments further proved that the type A CTD protein cellulase CHU1336 failed to anchor on the outer membrane of the deletion strains and was secreted to the outside of the cell in large quantities.The localization of type B CTD protein（pfam13585）CHU3220 in WT and deletion strains was no change.The above results all indicate that LPS plays an important role in the anchoring of T9SS cargo proteins on the cell surface.For the first time we revealed why LPS affects the cellulose degradation ability of C.hutchinsonii.In addition,the apparent molecular weight of some outer membrane proteins in WT is larger than that of the deletion strains.To investigate the reason,one of the differential proteins CHU0125 was selected to express in situ.CHU0125 predicted the molecular weight of the mature protein to be 70 kDa.Western blot results showed that there are two bands,about 70 kDa and 85 kDa on the periplasmic space,and the band on the outer membrane is about 100 kDa,indicating that CHU0125 has modifications in both the periplasmic space and the outer membrane,causing its apparent molecular weight to increase.The endoglycosidase PNGase F can cleave the glycans attached to the protein Asn site and cause the molecular weight difference.When the protein 0125-FLAG-tag is treated with PNGase F,there is no difference in molecular weight.It is speculated that the modification of CHU 0125 is not N-Glycosylation modification,and the modification on the outer membrane may be caused by LPS,which needs further verification.The periodic acid oxidation method was applied to stain glycoproteins,it was found that there were a large number of glycosylated proteins in the periplasm and outer membrane of WT.However,there are only a few visible glycoproteins inΔ2777 and Δ2176,and a large number of protein glycosylation modifications have disappeared,further indicating that LPS synthesis-related genes may be involved in the glycosylation modification process of protein in C.hutchinsonii,its modification process,method,physiological and biochemical significance need to be further explored.At the same time,the ability of Δ2777 and Δ2176 to form biofilms and the resistance to adversity are reduced,which may be related to the loss of O-antigen destroying the integrity of the outer membrane.Functions study of glycosyltransferase CHU 1044:Insertion mutation screened to a gene chu1044,encoding b-glycosyltransferase,glycosyltransferase 2 family protein.chu1044 is located in the inner cell membrane and has 7 transmembrane domains.It has 58.7%amino acid sequence identity with the glycosyltransferase gtfC of Porphyromonas gingivalis,gtfC was involved in the A-LPS synthesis.Deletion of chu1044 results in the loss of O-antigen,the bacteria cannot degrade filter paper,the gliding ability on soft and hard agar was lost,and it cannot be arranged on the surface of filter paper fiber as neatly as WT.The cell surface endocellulase activity of Δ1044 is almost completely lost,while the whole cell endocellulase activity decreases by more than 50%in the early growth period,theβ-glucosidase enzyme activity on the cell surface and the whole cell is also significant decline in the early growth period.Analysis of cellulase activity by renaturation electrophoresis showed that the distribution of cellulase in the periplasm and outer membrane of Δ1044 was significantly less than that of WT,while the abundance of extracellular cellulase was higher than WT.We used proteomics technology to identify WT and Δ1044 cell surface proteins.Only 14 A-type CTD proteins were identified on the cell surface of Δ1044,while 47 were identified on the surface of WT cells.This is consistent with the experimental results of Δ2777 and Δ2176.Of the 18 cellulases currently predicted,12 have A-type CTD sequences,which are substrate proteins of T9SS and play an important role in the cell surface cellulose degradation pathway of C.hutchinsonii.Therefore,it is speculated that the lack of LPS causes the type A CTD proteins cannot anchor on the cell surface is the mainreasonwhy Δ1044 cannot degrade cellulose.The outer membrane of Δ1044 also has a large number of proteins whose apparent molecular weight is smaller than WT.The process of glycosylation modification requires multiple glycosyltransferases.Different component proteins of WT and Δ1044 are extracted,and glycoproteins are stained by periodic acid oxidation.The experimental results show that the absence of chu 1044 does cause glycosylation of a large number of proteins.Loss of modification.Considering that chu 1044 also participates in the synthesis of LPS O-antigen,the glycosylation modification process involved in chu 1044 may be coupled with the synthesis process of O-antigen,which requires further experimental verification.The CTD protein in P.gingivalis is anchored to the cell surface by anionic lipopolysaccharide.There is currently no evidence to support the existence of A-LPS in C.hutchinsonii.Thro μgh preliminary studies on the functions of C.hutchinsonii LPS synthesis related genes O-antigen flipases chu2777,chu 2176 and glycosyltransferase chu1044,we speculate that the type A CTD in C.hutchinsonii is anchored to the cell surface by LPS.Different from the common insertion or complex anchoring of outer membrane proteins,this fully exposed anchoring is more conducive to the contact between cellulase and the substrate,conducive to rapid and efficient degradation.At the same time,the periplasmic and outer membrane protein glycosylation modification of C.hutchinsonii was preliminary explored.This provides us with a new idea for the unique cellulose degradation and sliding mechanism of C.hutchinsonii,and provides a reference for the modification and anchoring of the surface protein of the Bacteroides phylum microorganism. |
