Characterization Of ChuDegQ And Functional Study Of The Peptidoglycan Associated Lipoprotein In Cytophaga Hutchinsonii

Cytophaga hutchinsonii is an aerobic gram-negative bacterium from Bacteroidetes and it could degrade crystalline cellulose efficiently.However,C.hutchinsonii does not secrete free cellulase or produce cellulosome when degrading cellulose.Analysis of C.hutchinsonii genome revealed that C.hutchinsonii did not encode exocellulase and all the cellulases didnot contain carbohydrate binding module?CBM?.During cellulose degradation,C.hutchinsonii required contacting with cellulose directly and most of the cellulose activities were cell-surface associated,suggesting the important role of outer membrane proteins in cellulose degradation.Therefore,the researchers speculated that C.hutchinsonii may adopt a new and unknown mechanism to degrade cellulose.The degradation mechanism is roughly as follows:the cellulose adsorption proteins on the cell surfaces adsorb on cellulose,and then the cellulose chains are peeled by protein complexes and transported to the periplasmic space for degradation.During the entire cellulose degradation process,the outer membrane proteins and periplasmic space proteins are very important.DegQ,which could be found in many organisms,is an htrA?high temperature requirement A?family protein.It could control protein quality of the outer membrane and periplasmic space by degrading or refolding proteins misfolded proteins.The data from our laboratory indicated that deletion of DegQ resulted in C.hutchinsonii could not degrade crystalline cellulose,indicating the important role of DegQ in C.hutchinsonii.However,people know little about the characterization of DegQ in C.hutchinsonii and Bacteroidetes.In order to enrich people's understanding of DegQ in Bacteroidetes,Chu DegQ of C.hutchinsonii was heterologously expressesed in E.coli and the properties were studied.Although researchers speculated that the outer membrane proteins of C.hutchinsonii play an important role in the degradation of cellulose,in recent years only several outer membrane proteins such as CHU₁276,CHU 1277,CHU₀170,and CHU₃220 were identified to affect the cellulose degradation in C.hutchinsonii.In this paper,a new outer membrane protein,peptidoglycan-associated lipoprotein?Pal?,was found to affect the degradation of amorphous region of cellulose in C.hutchinsonii.Meanwhile,we also studied the gene function of Hp₄,which was located at the downstream of chu₁276 and chu₁277.The specific research content and the results obtained are as follows:1.Characterization of bifunctional protease-chaperone ChuDegQChuDegQ consisted of 483 amino acids and contains conserved chymotrypsin-type serine protease,PDZ1,and PDZ2 domains.The similarity to EcDegQ derived from Escherichia coli was with a sequence identity of 42%.EcDegQ is a bifunctional enzyme that has been reported to have both serine protease activity and molecular chaperone function.We expressed ChuDegQ inE.coli and obtained a large number of active proteins and examined the properties of ChuDegQ.The optimum reaction temperature of ChuDegQ was detected with BSA as substrate.The optimum reaction temperature was about 50?,and the optimum reaction pH was about 8.5.Using lysozyme as a substrate for the chaperone activity of ChuDegQ,it was found that ChuDegQ could prevent the aggregation of lysozyme.But as the incubation temperature increasing from 25?to 35 ?,the chaperone activity gradually weakened until disappeared.The ability of ChuDegQ to degrade denatured proteins and prevent aggregation of denatured lysozyne was similar to that of EcDegQ.We deduced that ChuDegQ is a typical bifunctional protease-chaperone protein.Further study of the protease properties of ChuDegQ revealed that it coud only degrade denatured BSA.When denatured BSA and nature BSA were mixed together,ChuDegQ could identify and degrade denatured BSA by a special recognition mechanism.Further investigation of the chaperone properties of ChuDegQ showed that ChuDegQ could prevent aggregation of denatured lysozymes,but ChuDegQ did not restore activity of lysozyme that had already aggregated.An important feature of DegQ is that it can form different polymerization forms such as trimers,12-mers,and 24-mers.For a long time,it has been considered that 12-mers and 24-mers are preconditions for DegQ to perform protease or chaperone function.Until 2012,Kim et al.found that the trimeric DegP?a-homolog of DegQ?had normal protease activity,but it is still unknown whether DegQ is.By adding different concentrations of denatured lysozyme,SEC detected the trimer and dodecamer of ChuDegQ.We obtained the trimer of ChuDegQ by controlling the concentration of lysozyme or by site-directed mutation.We tested the ability of the trimeric ChuDegQ to degrade denatured proteins and found that the trimer had the same activity as the dodecamer,which showed that the dodecamer of ChuDegQ is not necessary for the degradation protein.However,it came along with severe self-degradation when trimeric ChuDegQ degrade denatured proteins,which might be caused by the unstable form of the trimer.Trimeric ChuDegQ was found to prevent the aggregation of lysozyme,suggesting that trimeric ChuDegQ showed chaperone-like property.However,as the increasing of the concentration of lysozyme,the chaperone property of trimeric ChuDegQ was weaker than that of dodecamer.MalS was also used as the substrate to test the chaperone ability of trimer and dodecamer and the result suggested that the chaperone ability of trimer was about 35%of that of dodecamer.Thus,we found that the trimeric ChuDegQ can both prevent the aggregation of denatured lysozyme and also restore the vitality of MalS,so the trimeric ChuDegQ could function as a chaperone.This is the first report about the protein properties of DegQ derived from Bacteroidetes,which enriched the understanding of the htrA protein from Bacteroidetes.Meanwhile,we found that the trimeric ChuDegQ possessed protease activity and chaperone function,which was the comprehensive report of a trimeric DegQ with protease activity and molecular chaperone function.Compared to the trimeric form of ChuDegQ,the 12-mer form of ChuDegQ was resistant to self-degradation and showed a normal molecular chaperone function,which also provides a theoretical basis for explaining why DegQ forms dodecamer.2.Function of peptidoglycan associated lipoprotein and its effect on cellulose degradation in Cytophaga hutchinsonii.Pal encodes an outer membrane peptidoglycan associated lipoprotein?pal?in C.hutchinsonii.Bioinformatics analysis revealed that besides the conserved OmpA_C-like domain,ChuPal possessed another two TPR?tetratricopeptide repeats?and PD40 domains that Pal does not have reported.These two domains may confer different functions on ChuPal.The homologous recombination method was used to knock out Chupal and cellulose degradation ability of the mutant strain ?pal was detected.When glucose was used as the carbon resource,it was found that ?pal could grow normally in the inorganic medium Stainer,but Apal exhibited a long lag period and a reduced biomass at the stationary phase compared with the wild type.When PY6 medium was supplemented with 1 g/L N03-the Apal mutant could grow as well as the wild type,suggesting that the Apal mutant could not grow as well as the wild type in PY6 medium and additional N03 was important for the growth of the ?pal mutant.When amorphous cellulose?RAC?was used as the sole carbon source,the growth rate and final biomass of Apal were similar to thatof wild type,but ?pal could not grow normally when cellulose?Avicel?was used as the carbon resource.At the same time,we detected the utilization of cellulose and found that after culturing 51 h,the wild type could use 81%of Avicel and Apal could only use 12%of Avicel.These results indicated that the lack of Chupal affected the degradation of crystalline cellulose in C.hutchinsonii.We further measured the crystallinity of Avciel by X-ray diffraction?XRD?and found that the crystallinity of Avicel which was treated with the wild-type was reduced from 65.1%to 57.4%,but that of Apal increased from 65.1%to 70.7.%,indicating Apal mainly used the amorphous region of the Avicel.We also observed the arrangement of the bacteria on the filter paper with SEM and found that the wild type was arranged orderly on the surface of the fiber,but the Apal was only grown in the gully of the fiber,After washing out the bacteria,it was found that the surface of the wild-type degradation filter paper was smooth,but that of ?pal was gully-shaped.Combining the previous results,we speculated that this gully-shaped surface might be caused by the selective degradation of cellulose by the A pal mutant.The cellulase activity of C.hutchinsoniiwas also detected and we found that the endoglucanase and b-glucosidase activities on the cell surface were reduced by 60%and 30%without Chupal,respectively.But the intracellular cellulase activity was not affected at all,indicating that the deletion of Chupal only affected the cellulase activity on the cell surface and the surface of Apal might be damaged.We further tested the sensitivity of C.hutchinsonii to some toxic compounds and found that Apal was more sensitive to toxic compounds compared with the wild type.The amounts of some outer membrane proteins were remarkably decreased in the pal deletion mutant.We detected the outer membrane vesicles of C.hutchinsonii and found that the pal deletion mutant released six-fold more outer membrane vesicles?ONVs?whose protein varieties were dramatically increased.All the results showed that deletion of pal impaired outer membrane integrity of C.hutchinsonii.We further examined the localization of CHU—3220,a key protein that localized on the outer membrane and influenced the degradation of the crystalline region of cellulose.Western Blot showed that the amount of CHU₃220 on the outer membrane of ?pal was reduced by nearly 75%.These CHU₃220 leaked from the outer membrane were almost stored in outer membrane vesicles and might not perform their functions normally.This work revealed that deletion of pal which encoded a putative outer membrane peptidoglycan-associated lipoprotein prevented degradation of the crystalline region of cellulose,which improved our understanding of the function of Pal.This study also enhanced the importance of the integrity of outer membrane in the degradation of the crystalline region of cellulose by C.hutchinsonii.3.Functional study of a hypothetical protein in C.hutchinsonii Hp₄ was located at the downstream of Hp₁,Hp₂ and Hp₃.In order to study the gene function of Hp₄,we knocked out Hp₄ through homologous recombination and found that ?Hp₄ could normally slide on soft agar but dould not degrade filter paper,indicating that Hp₄ was also a key gene for cellulose degradation.We added 0.1%glucose to the Stainer plate and found that ?Hp₄ restored the ability to degrade cellulose.We analyzed the role of 0.1%glucose in the degradation x of cellulose.It was found that AHp₄ which was preclutured by 0.1%glucose medium still failed to degrade cellulose.However,AHp₄ restored the ability to degrade cellulose when enlarged the inoculant.Therefore,we speculated that 0.1%glucose might promote the initial growth and accumulation of AHp₄.The upstream genes Hp₁,Hp₂ and Hp₃ of Hp₄ are key genes for cellulose degradation in C.hutchinsonii.By bioinformatics analysis,we found that Hp₁-Hp₄ might be a conserved sequence in cellulose-degrading bacteria.The experimental results showed that by enlarged the inoculant,A Hp₁,A Hp₂,A Hp₃ restore the ability to degrade cellulose.Therefore,we speculate that Hp₁-Hp₄ plays a key role in the cellulose degradation of C.hutchinsonii and that deletion of these genes might affect the initial growth and accumulation of C.hutchinsonii.