Cloning The Strong Promoter And Research On The Functional Regions And Structure Properties For Cytophaga Hutchinsonii

The Cytophaga hutchinsonii(abbreviated as C.hutchinsonii)is a Gram-negative aerobic bacteria that belongs to the phylum of Bacteroidetes.They exhibit gliding motility on the soft agar or wet glass.They could degrade crystalline cellulose rapidly and efficiently.The mechanism for cellulose degradation activity and the gliding motility still remain unclear.The Chutchinsonii neither secrete free cellulases nor possess cellulosomes structure,indicating that they may employ a novel mechanism for cellulose degradation and motility.It has been demonstrated that the cellulose degradation process is always accompanied with their motility.The glide protein plays as the executor for Chutchinsonii motility.We failed to generate the recombinant glide protein from E.coli due to the formation of inactive inclusion body.The expression and secretion of proteins are usually controlled by the complex and sophisticated regulation mechanisms which including many regulation motifs,such as promoters,enhancers,attenuators as well as other cis-acting elements.At present,little knowledge has been known on the promoters of Bacteroidetes species.Thus it is particularly important to identify conserved sequences of promoters and internal regulatory elements from different Bacteroides,such as determination of the conserved sequences of promoters,the position of the transcription start site(TSS),and specific up-regulation elements or down-regulation elements.According to the analysis of the transcriptome data of the C.hutchinsonii,we screened a number of highly transcripted genes.We chose their promoters as targets for our study.We used these promoters to construct a series of expression vectors by using lacZ reporter gene and evaluated the promoter activity by measuring the enzyme activity of LacZ.Finally,we obtained five strong promoters:Pchu_fanA,Pchu_fanB,Pchu_fanC,Pchu_fanC and Pchu_fanE for the C.hutchinsonii.The transcription start site of the promoters in C.hutchinsonii were determined by 5'RACE(Rapid Amplification of cDNA Ends).We demonstrated that the promoters possess two conserved regions are-5 region(TAAT)and-31 region(TATTG)and the spacer length is 21 bp or 22bp between the two regions by comparing the upstream sequence of the transcription start site.Subsequently,mutagenesis study on the promoter Pchu_fanA was conducted using site-directed ligase-independent mutagenesis(SLIM).The results shows that when the base in the sites of-31,-32,-33 for the first conserved region was mutated,the promoter activity decreased significantly.The bases at the sites of-34 and-35 did not affect the promoter activity due to their lower conservation compared with the first conserved region for the promoter Pchu_fanA.When all the bases in the first conserved region of promoter Pchu_fanA had been mutated to their complementary bases,the activity of the promoter is almost diminished completely.And we found that the activity of the promoter decreased when mutated the bases to guanine or cytosine in the second conserved region of promoter Pchu_fanA while mutation of the second conserved region bases to adenine or thymine bases does not have a significant impact on promoter activity.In addition,the promoter activity was decreased after insertion or deletion the thymine bases for the spacer length between the two conserved regions,and the optimal spacer sequence was 22bp for the Pchu_fanA promoter of the wild-type C.hutchinsonii,indicating that the promoter of the C.hutchinsonii is very conservatism in evolution process.During the process of screening the promoter,we found the strong promoter always accompany with a numbers of TTTG motifs.Therefore,we inserted the different copies of TTTG sequences into the two weak promoters Pchu_fanG and Pchu_fanF of the C.hutchinsonii.The results shows that when 12 and 24 copies of TTTG sequences were inserted into the promoter Pchu_fanG and Pchu_fanF the promoter strength is 31.1 and 46.9 times higher than the original level respectively.And we performed the TTTG sequence superpose deletion experiment for the strong promoter Pchu_fan4 at the sites of-379,-314,-203 and-189,and we found that the promoter activities was lower than the original level has been decreased by 25.9%,29.6%,63.0%and 74.1%respectively.It was demonstrated that there was a positive correlation between the numbers of TTTG sequences and promoter activities.On the other hand,in silico analysis on the genome of C.hutchinsonii and other Bacteroides species demonstrated that 61.6%genes in genome exhibit the same-5 and-31 conserved region in all of promoters for C.hutchinsonii by bioinformatics.There are 26.9%genes in genome have been identified with this conserved promoter structure in the genomes for Flavobacteria johnsoniae.However,only 2.4%genes in genome with this conserved structure in coli could be retrieved,indicating that the phylogenetic relationship may determine the similarity of conserved promoter structure in different species.In conclusion,in depth investigation on the promoter structure of C.hutchinsonii not only fill the blank space of our understanding of the promoter structure,but also establish a good foundation for further exploration of the special mechanism of cellulose degradation by the Chutchinsonii,as well as act as an important supplement for recognition the promoter structure of phylum Bacteroidetes microorganisms.