Study On The Relevant Genes In Cellulose Utilization And Gliding Motility Of Cytophaga Hutchinsonii

With oil, coal and other non-renewable resources gradually depleting, exploring renewable energy utilization has become an urgent need. As the main component of plant biomass, cellulose is the most abundant renewable resource on earth. Degradation of crystalline cellulose is the key factor for lignocellulosic biomass conversion. In addition to the free cellulase system of fungi and complexed cellulosome of anaerobic bacteria, some aerobic bacteria such as Cytophaga hutchinsonii have a strong ability to degrade crystalline cellulose in a different manner. Genome sequences of C. hutchinsonii suggested that it do not have cellobiohydrolases and endoglucanases lack the cellulose binding domain, genes encoding typical motility organelles are also absent.Preliminary results indicated that C. hutchinsonii degrade crystalline cellulose depending on its contact to the subsrate, and the cellulase activities mostly located in the cell surface. Besides, the uptake and utilization of cello-oligosaccharide may constitute an important step of the cellulose degradation process. C. hutchinsonii can glide quickly along the surface of solid medium. Inactivation of the gliding motility gene gldJ led to the loss of cell motility as well as the ability of cellulose degradation. It was inferred that there may be a link between cellulose degradation and cell motility. Since cellulases are always difficult to be heterologously expressed with activities, it’s urgent to establish an efficient genetic manipulation system so that genes involved in cellulose degradation can be efficiently identified. This thesis explored the genetic manipulation tools of C.hutchinsoni and initially established the genetic manipulation system; on the basis of a lot of transposon screening, some cellulose-utilization deficient mutants were gained; according to the bioinformatic analysis, a hypothetical model of cellooligosaccharides transport by C. hutchinsonii was proposed. The main contents and results are as follows:1. Cultivation of C. hutchinsonii on solid medium was optimized and genetic manipulation system was developed preliminarily.In order to establish the genetic manipulation system of C. hutchinsonii, cultivation of strains on solid culture was been promoted at first and we found that the appropriate agar concentration for colony formation was0.6%. Tolerance of different antibiotics for wild-type strain was explored and results indicated that erythromycin, chloramphenicol or tetracycline can be used as resistance selection markers for C. hutchinsonii. A variety of genetic transformation methods had been tried. The efficiency of electroporation was found relatively higher and more stable than that of conjunction. Then a replicating plasmid carrying the replication origin from C. hutchinsonii genome and a transpon plasmid were successfully transported into C. hutchinsonii. It was verified that these resistance gene can work in the strain and the appropriate screening concentrations were determined. Then a complement plamid with chloramphenicol resistance gene was constructed. Some promoters which could promote GFP expression in C. hutchinsonii can be used to express many other proteins in vivo in future.2. Inactivation mutant Chin1107, Chin1280and Chinl335were constructed by targeted gene insertion and it was indicated that inactivation of any one of cellulase genes may not decrease the ability of cellulose degradation of C. hutchinsonii.A single cellulase may play different roles in different bacteria. As is known, C. hutchinsonii has a unique cellulose degradation mechnism, wherein in order to study whether a critical component of the noncomplexed cellulase system is existed, we constructed insertion inactivation mutant strains of three cellulase genes, respectively. The growth of Chin1107, Chin1280and Chin1335were similar to wild type strain, no matter glucose or avicel as the sole carbon source in liquid medium. The difference was also not apparent when cultivated on filter paper plate. Perhaps the lack of a single cellulase did not seriously affect the cellulose utilization of C. hutchinsonii. Outer membrane, inner membrane and cytoplasmic proteins were seperated from wild-type cells incubated in PY10supplied with different carbon sources. Among all the proteins, one was induced in cellobiose compared to glucose. By mass spectrometry, it was identified as Bg1X which is located in the periplasmic and annotated as β-glucosidase. The transcriptional of bglx gene was upregulated in cellulose cultures. The β-glucosidase Bg1X may play an important role in utilization of cellobiose and cellulose. 3. A mutant deficient in cellulose degradation was obtained by transposon mutagenesis screening. The ability of colony spreading and components of outer membrane proteins were both different between the mutant and wild type strains.A transposon mutant ChT68was obtained with the phenotype of nomal growth in liquid medium but weaker growth on solid medium with cellulose as the sole carbon source compared to the wild type. It was identified that the insertion position of transposon was chu₁719encoding a hypothetical protein. Targeted gene inactivation of chu₁719was achieved by electroporation of suicide vector pLYIN1719. The inactivation muant Chin1719exhibited same phenotypes with ChT68. Colony spread of Chin1719was deficient on hard agar plate. CMCase activity of membrane proteins was reduced by about40%than the wild type and cellulose adhesion proteins from outer membrane proteins of Chin1719and WT were significant different. Differential proteins included G1dJ, G1dN, CHU₀007, CHU₃434and CHU₃732. These results suggested that CHU₁719may play a role in cellulose utilization, spreading and protein secretion.4. Inactivation of chu₀546and chu₀553which were annotated as susC-like genes did not influence cellulose utilization of mutant strains. However, a predicted TonB-dependent receptor-encoding gene, chu₁276, was considered to play an important role in cellulose utilization by constituting the sus-like system with its downstream genes.As is known, a cell envelope-associated multiprotein system named Sus （starch utilization system） enables Bacteroides thetaiotaomicron to bind and degrade starch, of which SusC and SusD are essential components. Chin0546and Chin0553, of which susC-like genes chu₀546and chu₀553was inactivated respectively, grew normally on glucose and cellulse. T127, T423and C-34, the transpons mutants of chu₁276, chu₁277and chu₁278could not degrate cellulose. A quite long lag phase can be observed when T127was cultivated on liquid medium with0.1%glucose or cellobiose as the sole carbon source. Upstream genes chu₁265/chu₁266was also inactivated, but no differential phenotypes between mutants and WT were detected. CHU₁276was located in outer membrane and could respond to low concentration glucose. As a DNA-binding module was also predicted at its C-terminal, we speculated that CHU₁276was not only a SusC-like protein but also a transcription regulator. The transcription of some cellulases was down regulated in T127correspondingly. CHU₁278should be located to lipid bilayer of outer membrane to function normally.5. The gene expression differentiation was evaluated by transcriptome analysis between wild-type and inactivated mutant of chu₁276on different carbon sources. Several genes including those respond to cellulase inducing signals, involved in metabolic pathway and signal transduction were identified. These results supplied us with more details about the transcriptional regulation of cellulase in C. hutchinsonii.Total RNA of wild-type and T127were extracted from similarly induction conditions with different carbon sources. mRNA sequence were separated and sequenced. The data were analyzed and classified giving the following results:Compared to the non-carbon source condition, there were up-regulated genes and down-regulated genes both when cultured on glucose and cellulose. Among them, genes encoding hypothetical proteins accounting for a large part. Through GO and Pathway significant enrichment analysis, no processes involved in carbohydrate metabolism were figured out. In wild type strain, the most endoglucanases can be induced in non-carbon condition, but not specifically cellulose induced; while3β-glucosidases were specifically induced by cellulose. The expression of chu₁276～chu₁279was cellulose-specific induced, and their transcription levels in T127were down-regulated. So it is inferred that the chu₁276～chu₁279loci may play an important role in the cellulose degradation of C. hutchinsonii.