Functional Study Of Genes Related To Protein Folding And Location In Cytophaga Hutchinsonii

Cytophaga hutchinsonii is a widely spread aerobic cellulolytic bacterium in soil,which belongs to the phylum Bacteroidetes.Different from the known cellulase synergistic degradation mechanism and fibrosomal degradation mechanism,C.hutchinsonii utilizes a novel and unclear mechanism to degrade cellulose.Direct contact with cellulose is necessary for cellulose degradation in C.hutchinsonii.No predicted exoglucanase was found,and most of cellulase activities are cell associated.In addition,it exhibits gliding motility over surface,while the mystery of gliding motility is also unknown since it has no flagella or pili.Previous studies in our lab found that C.hutchinsonii had two ways to degrade cellulose in cell surface and in the periplasm,and the outer membrane proteins play essential roles in cellulose binding and degradation in cell surface.Therefore,appropriate folding in the periplasm,secretion and location in the outer membrane are important for the function of proteins.Disulfide oxidoreductase catalyzes the formation of disulfide bonds between cysteine residues of proteins,which plays an important role in stabilizing the spatial structure and function of proteins.Disulfide oxidoreductase is rarely reported in Bacteroidetes.Previous study found that a hypothetical protein CHU 1165 in C.hutchinsonii showed 32%identity with TlpB,a possible disulfide oxidoreductase in Flavobacterium psychrophilum which also belonged to the phylum Bacteroidetes.Deletion of CHU 1165 caused cellulolytic defect and gliding defect,indicating that disulfide oxidoreductase might be involved in folding the proteins that were related to cellulose degradation and gliding motility.In addition,type ? secretion system?T9SS?is unique in the phylum Bacteroidetes,and C.hutchinsonii contains all of the T9SS homologous.Previous studies found that some components of T9SS in C.hutchinsonii affected cellulose degradation and cell movement,while the functions of other T9SS components were unclear.In this study,we deleted the homologous gene of T9SS-related gene porV,chu₃238,and verified its function to increase the understanding of T9SS in C.hutchinsonii.The main research contents are as follows:Identification of disulfide oxidoreductase CHU₁165 and its functional study in C.hutchinsonii.Bioinformatic analysis showed that the N-terminal region of CHU₁165 has five predicted transmembrane domains,and the C-terminal region stretches to the periplasmic space which contains a C266XXC269 motif that is conserved in disulfide oxidoreductases.In addition,the C106XC108 in the N-terminal transmembrane region is also conserved in TlpB.We mutated all 12 cysteines of CHU₁165,and found that the mutation of the four conserved cysteines in C266XXC269 and C106XC108 resulted cellulolytic defect in the mutants,suggesting that they were essential for the function of CHU₁165.CHU₁165 expressed in the periplasm of E.coli BL21 was oxidized and in the cytoplasm of E.coli BL21 was reduced,indicating that CHU₁165 could form disulfide bond and both of them showed disulfide oxidoreductase activity since it could reduce insulin in vitro.Western blot showed that CHU₁165 was at the inner membrane and the CXXC motif was in an oxidized state,indicating that CHU₁165 was a membrane-anchored disulfide oxidase in C.hutchinsonii.CHU₁165 was the first disulfide oxidoreductase verified in C.hutchinsonii.In the deletion mutant of CHU₁165,?1165,the intact cells displayed a 2-fold reduction of endoglucanase activity compared to the wild type,while lysed cells showed a 30%reduction in endoglucanase activity compared to the wild type?average from three growing phases?.The ?-glucosidase activity of intact cells and lysed cells of the A1165 mutant was reduced about 25%from that of the wild type.The deletion of chu₁165 affected cellulase activities,while the ?1165 mutant retained the most of cellulase activities,which would not lead to the absolute defect in cellulose degradation.The first step for C.hutchinsonii to degrade cellulose is to bind cellulose,and the outer membrane proteins play important roles in cellulose binding.The ?1165 mutant cells could hardly bind to cellulose,and the outer membrane proteins capable of binding to cellulose in the ?1165 mutant were obviously decreased.This is the main reason for cellulolytic defect in the ?1165 mutant.Many decreased proteins in the ?1165 mutant were involved in cellulose degradation and gliding,however,these proteins contained no or one cysteine.The mutation of cysteine in these proteins did not affect cellulose degradation,indicating that these proteins did not form disulfide bond.CHU₁165 may have other way to affect the function of these outer membrane proteins,which increased the understanding of disulfide oxidoreductase in the phylum Bacteroidetes.Functional study of T9SS-related gene chu 3238 in C.hutchinsonii.The substrates of the T9SS contain the conserved C-terminal domain?CTD?.In Porphyromonas gingivalis,PorV is the outer membrane receptor of CTD signal peptidase PorU,and also involves in secretion and location of T9SS substrates in the outer membrane.CHU₃238 exhibits 38.6%identity with PorV,which may be the component of the T9SS.Deletion of chu 3238 caused cellulolytic and gliding defects in the A3238 mutant.In terms of growth,the A3238 mutant showed a significant growth defect in organic medium PY6,and the final biomass is only one-third of the wild type.When the Stanier medium containing five metal ions was added,the growth was normal,suggesting that CHU₃238 may be involved in metal ion absorption,which is similar with the component of the T9SS,GldN in C.hutchinsonii,and this function has not been found in the T9SS of other Bacteroidetes.In terms of protein secretion,a 70%reduction in endoglucanase activity and 42%reduction in ?-glucosidase activity of intact cells in the ?3238 mutant compared to the wild type.Twelve predicted cell-surface endoglucanases contain the CTDs in C.hutchinsonii,indicating that an incomplete T9SS possibly caused the failed secretion of cellulases in the ?3238 mutant.In addition,deletion of PorV in P.gingivalis caused CTD proteins to be accumulated in the periplasm.In the A3238 mutant,the outer membrane and extracellular proteins were significantly decreased compared to the wild type,but the periplasmic proteins did not show the difference from the wild type.We found that the transcription of periplasmic protease DegQ was 1-fold higher in the ?3238 mutant than the wild type,suggesting that the overexpressed DegQ degrade those unsecreted proteins to maintain the balance of periplasmic proteins.Western blot showed that CHU₃238 was in the outer membrane,and the ?3238 mutant was more sensitive to toxic reagents compared to the wild type,indicating that the deletion of chu₃238 affected the outer membrane integrity.These results suggest chu₃238 is vital for protein secretion and location in C.hutchinsonii,which enriches the understanding of CHU 3238 in the T9SS,and the phenomenon of metal ion-dependent growth of the ?3238 mutant shows the functional diversity of the T9SS.The above research results show that disulfide oxidoreductase and the T9SS are essential in protein folding,secretion and location,which provide help to reveal the unique cellulose degradation and gliding mechanism of C.hutchinsonii.