Tn4351 Insertional Mutagenesis Of Cytophaga Hutchinsonii And Identification Of The Mutants

Cytophaga hutchinsonii is a cellulolytic gliding bacterium distributing widely on the land. Since it can degrade crystaline cellulose efficiently,the US Department of Energy(DOE) had listed it in the microbial genome research.Its whole genome was sequenced in 2005.Although Cytophaga hutchinsonii can grow well in barren surroundings with cellulose as the carbon sourse,it grews pooly on glucose agar-medium.So it is difficult to study the characteristics of the strain.In this paper several factors that influence the growth of Cytophaga hutchinsonii was studied and the genetic conjugation system of Cythophaga hutchinsonii was optimized According to our study,besides cellulose,the optimized carbon sourse in the growth medium of Cythophaga hutchinsonii was filtrated- or 115℃,30min autoclaved glucose. The result showed that the culture conditon of the bacteria was a key factor in conjugation.Logarithm Medium-term cells are used for conjugation.The efficiency of conjugation was about 1.5×10^-6when conjugation was performed at 28℃for 24 hours with TC buffer or normal saline washing cells.The new mode overcomes the limitation of conjugation on filter paper.A mutants' library was established with Tn4351's random inserted mutation.At present,the number of mutants was more than 1000.The high efficient decomposition of crystalline cellulose was an evidence character of Cythophaga hutchinsonii.A mixed carbon medium was developed according to the different phenomenon of the strain when grew on glucose and cellulose.The carbon sourse in the medium was 0.5%glucose and 1%CF-11.Mutants with non- or weak-decomposing capacity with cellulose could be distinguished by the size of transparence zone around the colonies.Besides,the additions of 0.5%glucose could accelerate the growth of mutants.The new method improves the efficiency of screening.We got two mutants(27 mutant and 31 mutant)that decomposed cellulose more slowly than Cytophaga hutchinsonii CMC enzyme activities of the strains and the time course of filter paper degradation were tested.The CMCase activity of No.27 mutant was decreased by 20.7%.And the time course of complete degradation offilter paper was a week more than the wild type Cytophaga hutchinsonii.The inserted gene of No.27 mutant encoded a protein with unknown function.The enzyme′s activity of No.31 mutant was decreased by 40.7%. The time course of complete degradation offilter paper was 13 days more than Cytophaga hutehinsonii.The inserted gene of No.31 mutant encoded a secreted protein with unknown function.Gliding is another important character of Cythophaga hutchinsonii.In the paper, non-glide mutants' were also screened.Two mutants(39 mutant and 212 mutant) withenhanced gliding motility were obtained.The cellulaseactivity of 39 mutant was increase about 25.08%.No.39 mutant is a transcription regulator inserted mutant analyzed by biology software.The gliding motility of 39 mutant was similar to the Stype's gliding of myxobacteria.No.212 mutant can decompose cellulose.212 mutant is a DNA and RNA Helicase inserted mutant.The gliding motility of No.212 mutant was similar to the A- type's gliding of myxobacteria.Both of the mutants could degrade cellulose more effectively than the wild type which showed that cellulose degrading ability had something to do with gliding mobility.The screening and study of the decomposition and gliding deficiency mutants would be valuable for us to understand the cellulolytic mechanism and gliding mechanism of Cytophaga hutchinsonii.This study will help us to utilize the cellulose resource in nature more effectively.