Functional Studies On The Histone-like Proteins HU And IHF Of Cytophaga Hutchinsonii

Cytophaga hutchinsonii belongs to the phylum Bacteroidetes,which is a kind of aerobic gram-negative bacteria.It can rapidly digest crystalline cellulose in a contact-dependent manner without free cellulases or cellulosomes,which was proved a new strategy for cellulose degradation.Recently,with the development of the genetic operation system of C.hutchinsonii,many proteins that located in out membrane or periplasm have been demonstrated to be essential or important for cellulose degradation by C.hutchinsonii.However,little.is known about the factors which regulate the expression of these proteins.In addition,the functions of the histone-like proteins in C.hutchinsonii have not been studied yet.In this dissertation,the functions of the histone-like proteins HU and IHF in morphology,cell motility,biofilm formation,and cellulose degradation by C.hutchinsonii were elaborated by studying the phenotypes of the gene-deleted mutants and the characterizations of the recombinant proteins.The specific contents and main results are as follows:Research on the functions of the histone-like protein HU in C.hutchinsonii.HU is one of the most conserved and abundant nucleoid-associated proteins in eubacteria.This protein binds DNA in a sequence-independent manner and has anarchitectural role in DNA bending and compaction.Many HUs were reported to act as an important transcriptional regulator by combining with DNA.CHU₂750 is annotated as histone-like protein HUP in NCBI and mainly exists as a homodimer in C.hutchinsonii.The subcellular localization showed that CHU₂750 was in the center of the cell.The transcriptional analysis of chu₂750 by real-time quantitative PCR?qPCR?indicated that the expression of this gene in the wild type strain was significantly increased under the induction of crystalline cellulose,but not induced by cellobiose or amorphous cellulose,which suggested that CHU₂750 might be related to the degradation of crystalline cellulose.To elucidate the gene's function,the chu₂750-deleted mutant??2750?was constructed.The results showed that,although?2750 could utilize amorphous cellulose,it exhibited obvious defects in the degradation of crystalline cellulose,cell motility,and biofilm formation.Further studies revealed that A2750 displayed significantly decreased cell surface and intracellular endoglucanase activities but with ?-glucosidase activities similar to the wild type strain.Moreover,A2750 yielded much less exopolysaccharide than the wild type strain.The analysis by qPCR showed that the transcription levels of many genes involved in cellulose degradation and/or cell motility,including chu₃220,were significantly downregulated in A2750.CHU₃220 is a large protein,which is essential for the degradation of crystalline region but not amorphous region of cellulose by C.hutchinsonii.The fact that chu 3220 is significantly downregulated in A2750 may partially account for the compromised ability of A2750 in degrading crystalline cellulose.Our findings above demonstrated that,although chu₂750 is not an indispensible gene for C.hutchinsonii to survive,its deletion inhibits the division of cells,resulting in abnormal cell morphology and nucleoid structure.CHU 2750 is important for cellulose degradation,cell motility,and biofilm formation of C.hutchinsonii by modulating the transcription of certain related genes.Research on the functions of the histone-like protein IHF in C.hutchinsonii.The other protein which has a high identity of amino acid sequence with CHU 2750 in C.hutchinsonii is CHU₃687.Sequence alignment indicates that CHU₃687 is a homologous protein of the integration host factor?IHF?.The chu₃687-deleted mutant?A3687?was constructed to study whether this gene also plays a role in cellulose degradation like chu 2750.First,the results showed that the deletion of chu 3687 hampered not only the utilization of cellobiose or cellulose as the sole carbon source,but also the growth of C.hutchinsonii under the condition of inorganic nutritients.Then,the outer membrane proteins of ?3687 were compared with those of the wild type strain.It was found that the protein band of CHU 1277 was obviously absent in ?3687.CHU 1277 is an identified outer membrane protein which is essential for utilization of cellobiose and cellulose by C.hutchinsonii.Further research revealed that the recombinant CHU 3687 binded with the promoter region of chu₁277 by a DNA sequence of-250 to-550 bp upstream of the open reading frame and regulated the expression of chu₁277 in the process of transcription.The findings above indicated that CHU₃687 played a role in utilization of cellulose by modulating the transcription of the gene related to cellulose degradation.In this dissertation,the important role of histone-like proteins HU and IHF in cellulose degradation was determined,which provided help to reveal the unique mechanism of cellulose utilization by C.hutchinsonii and more functions in the histone-like proteins of Bacteroidetes.