Functional Study Of Chu₀922 And Chu₂830 Related To Cellulose Degradation In Cytophaga Hutchinsonii

As a kind of clean energy with abundant output,cellulose has been used as an alternative resource for fossil energy such as oil by more and more country.Microorganisms play a vital function in the degradation and utilization of fibrin Crystalline and amorphous structures are the main components of cellulose,and the crystalline part is very inseparable attributed to the intermolecular forces so that it is hardly degraded by microorganisms.Microorganisms degrade cellulose by two mechanisms.Anaerobes degrade cellulose by complexed cellulase systems and aerobic cellulose degraders utilize cellulose through the production of substantial amounts of extracellular cellulase enzymes.Cytophaga hutchinsonii is a widely distributed gram-negative bacterium which can quickly and efficaciously degrade cellulose and it is part of Bacteroidetes.The special things are that that C.hutchinsonii does not encode endocellulase and its endocellulase does not have the CBM domain.In addition,the analysis of the genome revealed that there was no cellulosomes structure in C.hutchinsonii.It relies on directly contact with insoluble cellulose to degrade cellulose.Therefore,it is speculated that there may be a third degradation strategy which the specific function of the strategy remains unclear.After several years of research,with the genetic manipulation technology of C.hutchinsonii improved,many important functional genes have been discovered and studied successively,which is enriched people's understanding of C.hutchinsonii.In this paper,two genes related to cellulose degradation were identified by transposon insertion mutation and been further studied.1.Functional study of chu 0922.In the process of screening for functional genes by transposon insertion mutation,a strain with defective degradation of filter paper was found.The insertion site of the mutant was identified as chu 0922 by reverse PCR.The phenotype of mutant strain obtained by knocking out the chu 0922 with the method of homologous double-crossover deletion is consistent with the transposon-mutagenized mutation,which cannot degrade cellulose.RT-PCR confirmed that the deletion of the chu 0922 did not affect the normal transcription of its upstream and downstream genes,which proved that the phenotype was indeed caused by the deletion of chu 0922.CHU₀922 is a hypothetical protein containing a signal peptide and Por Secre tail which is identified by T9SS.It is a typical T9SS substrate protein that is secreted extracellular via T9SS.The growth study of the ?0922 mutant under different carbon sources showed that the mutant could grow normally in glucose and cellobiose,however,the mutant in RAC had a 72 h lag phase and did not grow in Avicel.These results revealed that the A0922 cannot normally utilize amorphous cellulose and crystalline cellulose.Observing the sliding ability of the mutant strain on the surface of PY2,it was found that the deletion of chu 0922 prevents the sliding of individual bacteria and the diffusion of colonies from proceeding normally.The cellulosic materials treated by C.hutchinsonii was observed by scanning electron microscopy?SEM?The surface of the cellulose after treatment by WT was neat and smooth,and the surface of the cellulose treated by the mutant was rough,which is almost the same as the surface of the native cellulose.The crystallinity of the cellulose sample was detected by XRD,and it was found that the cellulose crystallinity which treated by WT strain was reduced,while the degree of crystallinity was raised when the cellulose were dealt with by the mutant strain A0922.This indicates that the mutant strain mainly utilizes the amorphous region of cellulose and cannot utilize the crystalline region of cellulose.Mass spectrometry was used to discern the differential proteins in outer membrane between WT and mutant strain A0922.It was found that the deletion of chu 0922 resulted in decreased expression levels of some outer membrane proteins,such as CHU 1276,CHU 1277,and CHU 1279.The mRNA of the mutant strain was extracted to detect the transcription level of chu 1276-1279 by RT-qPCR.It was found that the transcription levels of chu 1276,chu 1277,chu₁278 and chu₁279 were significantly lower in the mutant strain A0922 than WT,and the the down-regulation trend of chu 1276 and chu 1277 was more obvious when cultured with RAC as sole carbon source.Previous studies have shown that disruption of CHU 1276 in C.hutchinsonii resulted in complete deficiency in cellulose degradation,as well as compromised assimilation of cellobiose or glucose at a low concentration.CHU 1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization.These are vital genes for C.hutchinsonii to function normally.However,complementation any of the genes in chu 1276-1279 alone cannot restore the mutant strain to the wild-type filter paper degradation ability.C.hutchinsonii were fostered with gradient glucose substrate.The growth curve demonstrated that the slope and maximum of A0922 curve was raised with the increase of glucose concentration,but the growth rate of WT was the same under different glucose conditions.After that we examined the glucose uptake of cells in resting conditions and observed that the mutant strain A0922 had almost the same glucose absorption with WT under 0.1%or 0.4%glucose media at resting state.The glucose remaining in the buffer hardly changes with time increasing.When put in 0.1%glucose to the Stanier medium and the filter paper degradation experiment was carried out.It was found that the mutant strain could make the filter paper yellow,but still could not degrade the crystalline cellulose2.Functional study of chu 2830.A strain with defective degradation of filter paper was found by transposon insertion mutation and the insertion site of the mutant was identified as chu 2830 by reverse PCR.CHU 2830 was identified as a hypothetical protein with a TPR repeat located in the plasma membrane of the cell.And it showed slower utilization of cellulose and defective gliding ability than WT when deletion it.Explored the growth trend of A2830 under different carbon sources showed that the growth ability of the mutant strain was not affected when glucose and cellobiose were used as the sole carbon source,and the growth rate and maximum biomass were even higher than that of the wild type strain.However,when amorphous cellulose and crystalline cellulose were used as the sole carbon sources,the growth of the mutant strain has an obviously lag period,resulting in a significantly slower utilization of cellulose.It was found that the deletion of chu 2830 had almost no effect on the enzyme activity by detecting the endocellulase and ?-glucosidase activity on the surface and intact cells of the mutant strain.Finally,it turned out that the lag phase turned to normal when induction of?2830 by RAC and Avicel.