Screening Of Mutants Involved In Cellulose Digestion And Study On Chu₀602 Of Cytophaga Hutchinsonii

With the development of the society, people have realized that the problems such as lack of energy, environmental pollution bring a huge challenge to our society. In this situation, the technologies’exploitation of green energy must be a effective method for tackling this challenge. Lignocellulose producted by green plants is considered to be one of the lagest number of green energy in the earth, that has a broad application prospect. Due to the evolution of plants’ self-protection, in the natural condition cellulose always has a dense crystalline region, the crystalline region makes it is very hard to get the ability that can degrade cellulose as efficient as using cellulose microorganism with the existing technology, so it’s very necessary to know the mechanism of microrganism using cellulose in natural condition.Cytophaga hutchinsonii（C.hut） is a aerobic gram-negative bacteria, it belongs to the Bacteroidetes, which widely distributed in nature, it is different with all the cellulose degradation mechanisms that we have known. Cytophaga hutchinsonii has a extremely strong crystalline cellulose degradation ability. Cytophaga hutchinsonii can quickly glading on the surface of wet agar or on the surface of glass without dependent on the flagella or cilia; Cytophaga hutchinsonii neither secrete dissociative cellulose enzyme nor has a obvious cellulosomes structure, but it can effectively degrade crystalline cellulose and the mechanism of degradation cellulose and movement is not clear. When Cytophaga hutchinsonii degrade the crystalline cellulose, bacteria arranged on the cellulose fiber orderly, therefore some scholars believe that the cellulose degradation benefit from it’s movement, and it may be a novel mechanism.In order to get the genes associated with cellulose degradation, we adopted the approach of transposon insertion mutation by artificially induced. Transposon mutagenesis are adopted Tn4351 transposon, compared with the lab once have used HimarEm3, Tn4351 can insert more locus in the genome in bacteria, so the mutation source more widely. Tn4351 transposons are transformed into Cytophaga hutchinsonii by electroporation, then, transposons integrated into the bacterial genome by random insertion, and influence the expression of genes, affecting the phenotype of the bacteria. By inoculate mutons on the double cellulose medium tablet, we screened strains associated with cellulose hydrolysis degradation according to the status of cellulose degration by transformants. Cytophaga hutchinsonii has sequenced whole genome in 2007, and combined with bioinformatics to determine valuable genes for further research. We pick about 11000 transformants on the cellulose double-layer tablets, about 180 transformants have a obvious phenotypic, we selected 36 mutant strains with special phenotype,and molecular identification shows there are 36 locus （some genes have be screened repeatedly） in different strains including some important genes previously have reported chu₀134, chu₀170, chu₁075, chu₁798 etc. they have seriously affected the bacterial growth and cellulose degradation. Due to Tn4351 is always multi-insertion, which can get more insertion site, but multi-insertion make us couldn’t sure which one or which a few genes change eventually result in the phenotypic change, so it need gene knockout to research further. Comprehensive analyzed the gene we have screened except the important genes have been reported, we successively knockout some genes by homologous recombination.When screening mutant phenotypes we noticed that exclude several important genes which has been reported, and a pigment gene deletion mutant strain, chu 0602 gene deletion strain is one of the most obvious phenotype mutant strains, the ability of degradation cellulose is reduced, with the success of chu₀602 knockout, we found that the phenotype is consistent, which proves that lack of chu 0602 affected the cellulose degradation. NCBI notes that chu₀602 gene encoding lipid A core-O antigen ligase （Wzy_C）, speculative it played a key role in the process of lipopolysaccharides’（LPS） synthesis, chu₀602 deletion lead to lipopolysaccharide defection, which lead to defection in many respects, such as growth, cellulose degradation, movement and stress resistance etc. we cultivate mutant strains on stanier cellulose solid medium and Stanier filter solid mediumt, the strains lack of chu 0602 have a smaller hydrolysis circle than the wild type stains in same time. Mutant strains has a decreasing ability in movement on the soft and hard agar, this shows that lacking of LPS has an effect on individual and group movement. We have observed the mutant by optical microscope and scanning electron microscope （SEM）, mutant strains longer than the WT strains, especially at the adaptive phase or early logarithmic phase this kind of phenomenon is more obvious, Mutant strains on the filter paper looks more unordered and the surface looks more smooth than the wild type when growth in the filter paper。Growth curve measurement results show’s that compared use Avicel as the only carbon source with use the glucose as the only carbon source the mutant strains has a delay demurrage growth, and growth rate is lower then wild tipy strains. We tested the distribution of endo-cellulase activity andβ-glycosidase activity under the condition of different carbon source culture, found the new phenomenon that lack of lipopolysaccharide affect the distribution of endo-cellulase activity, but lack of lipopolysaccharide were not significantly affect the distribution of β-glycosidase activity. With glucose as the only carbon source, compared with wild strains, mutant strains endo-cellulase activity decreased obviously, In proportion to the surface of the cellulase activity of whole cell cellulase activity also declined obviously, The intracellular cellulase activity level a equate with the wild strains.Speculation that due to the lack of outer membrane lipopolysaccharide, affected the cellulase anchor on the surface of the cell.When use the Avicel as the only carbon source, the surface cellulase of mutant strains equate with wild strains, but the cellulase of the surface accounted for the proportion of whole cell drops greatly. Compared with the wild strains, mutant strains accumulated a large number of cellulase out of the cell. Speculation due to the lack of outer polysaccharide has affected the cellulase anchor on the surface of the cell, thereby released into the extracellular environment. Explain lipopolysaccharide is closely related to the cellulase anchor on cell surface. In the subsequent resistance test we found the mutant’s resistance become lower than wild type in the external environment pressure （such as SDS, antibiotics, H2O2, crystal violet, etc.）. SDS-PAGE shows the outer membrane protein, outer membrane attachment protein and exocytosis protein are all great changes compared chu₀602 mutant strain to the WT strain,The outer membrane protein and outer membrane cellulose adsorption protein decreased significantly, but extracellular protein increased significantly. We analyses the mass spectrum of the outer membrane protein which obviously change, many deleted outer membrane proteins associated with cellulose’s adsorption and the movement of bacteria. Speculated that because of the part lacking of LPS, influenced the integrity of the cell’s outer membrane and result in part of the outer membrane protein is released into the extracellular, which affect the bacterial cellulose degradation ability, etc.