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Isolation of Adherence-Defective Mutants and Research of Related Cellulose Binding Membrane Proteins

Author: LiHuiHui
Tutor: LiuWeiFeng;ChenGuanJun
School: Shandong University
Course: Microbiology
Keywords: Cellulose Mutants Membrane proteins Cellulase Adherence Mobility Two Dimensional Electrophoresis(IEF/SDS-PAGE) iTRAQ technology
CLC: Q933
Type: Master's thesis
Year: 2011
Downloads: 44
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Along with progress of society and the development of economy, the human beings have to be confront with many challenges, such as meager resources and shortage of energy, which is the undoubted barriers of society and economy. On the other hand, as the most abound renewable resource on the earth, cellulose would be brought new fundamental resources for society and economy. More than 1011 tons of cellulose was produced every year. However, because of its water-insolubility, high crystallization and strong force between cellulose chains(eg. hydrogen bond, hydrophobic interaction), biotransformation of cellulose is inefficient. Thus, it is one of the main concerns on the research of renewable resources to develop efficient methods of degradation of cellulose.In 1938, Cytophaga hutchinsonii, the abundant aerobic gram-negative celluloytic soil bacterium, was first isolated from the soil by Walker and Warren. Cytophaga hutchinsonii could degrade quickly the filters and posses the efficient capability of cellulose degradation. One research suggests that a third strategy to degrade cellulose is employed which is different from that of most aerobic cellulolytic microorganisms are employed by secreting a set of individual cellulases and that of most anaerobic microorganisms are used by cellulosomes complex. Similar to other cellulolytic bacteria, it is needed for Cytophaga hutchinsonii to direct contact with cellulose for efficient digestion, thus the adherence between strains and cellulose might be the first step to disgest the cellulose efficiently. Consequently, to research the mechanism of adhesion to cellulose would be benefit for understanding its unique strategy of cellulose degradation, which is crucial for the utilization of cellulose.In this thesis, we embarked on the isolation of adherence-defective mutants and their physiological and biochemical characterizations. The research was conducted as the following aspects. Firstly, isolation of mutants.To achieve C. hutchinsonii mutants, several mutant methods including UV mutagenesis, EMS mutagenesis, Spontaneous mutation and Transposon mutagenesis are employed. Two adherence-defective mutants, two gliding mutants and other unknown functional mutants of C. hutchinsonii were isolated.One adherence-defective mutant was isolated by employing the EMS mutagenesis and Spontaneous mutation, respectively. the capability of adherence was decreased steadily to 20-40%. Furthermore, the growth of these two adherence-defective mutants on glucose and cellulose were determined by measuring cellular proteins by digesting with NaOH. Whether growth on glucose or cellulose, the growth of mutants preformed weaker than that of wild type C. hutchinsonii, which is possible that the expression of some proteins such as the proteins related to transportation of oligosaccharides or proteins related to adhesion are changed or deleted in mutants.Based on the former researches, gliding along cellulose might be necessary for C. hutchinsonii to degrade the cellulose, which could enhance the ability of digestion of cellulose. Two mutants that whose spread capability on the agar medium were changed significantly, were isolated by employing UV mutagenesis. However, the growth of these two mutants kept in the same level with the wild type C. hutchinsonii.The further researches are needed to understand this phenomenon.Furthermore, several unknown functional mutants were also isolated through transposon mutagenesis. There are few change regardless of any aspects such as adhesion, mobility and growth. However, the inactivated genes were identified by plasmid rescue. The further researches of specific effects of these genes on C hutchinsonii are necessary.Secondly, isolation of membrane proteins.Nowadays, there is a trend that increasing researches focused on membrane proteins, including their structures, functions and generations. O Sequence analysis of genomes of Homo sapiens, Escherichia coli and Saccharomyces cerevisae suggests that about third of all genes of various organisms encode membrane proteins. However, due to their high hydrophobicity and low abundance, membrane proteins are demonstrated to be one of the most difficult researches. The former researches suggest that the adherence of C. hutchinsonii to cellulose might be related to the surface proteins of cells. Thus, the primary extraction and separation procedures of membrane proteins were developed in this thesis. We found that 1%DDM might be the ideal detergent to release the maximum membrane proteins being treated 3 hours at 4℃.Thirdly, identification of adherence membrane proteins through SDS/PAGEBecause of enrichment of adherence membrane proteins by binding to cellulose, they could be separated through the enrichment.. Six concentrated outer membrane proteins growth on glucose were detected by employing SDS/PAGE. Four proteins were identified by mass spectrum, of which three were hypothetical proteins, another One was related to mobility. The heterologous expression of this gliding motility-like protein was attempted yet failed, which was probably mainly due to the special structure of membrane proteins. Besides, the adherence membrane proteins of adherence-defective mutants and wild type C. hutchinsonii were also compared with similar method. Two more membrane proteins related to adherence were achieved.Fourthly, determination of differential membrane proteins under different cultural conditions through Two Dimensional Electrophoresis (IEF/SDS-PAGE)Cytophaga hutchinsonii requires direct contact with cellulose for efficient digestion, so some proteins might be over expressed on the cellulose. It is crucial for adherence membrane proteins through comparison of all membrane proteins and adherence membrane proteins when cells were cultivated on different carbon sources through Two Dimensional Electrophoresis (IEF/SDS-PAGE). In the comparison of differential proteins of all membrane, fourteen protein spots were detected, fourteen as well as in the comparison of adherence-related differential membrane proteins. The further researches about such as the identification of these proteins by MALDI-TOF MS and the characterizations of adherence membrane proteins are needed to explain the adherence mechanism.Fifthly, determination and analysis of differential membrane proteins on different carbon sources through Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)We determined the differential membrane proteins by comparison of all membrane proteins and adherence membrane proteins respectively growth on glucose and cellulose through Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). According to In the comparison of all differential membrane proteins on glucose and cellulose, amount about one hundred and eighty-two differential proteins were detected. Most of them were related to adherence and catalytic in molecular function. Among them, the expression level of ninety differential membrane proteins was up-regulated on cellulose.Besides, We also detected several enriched membrane proteins when binding to cellulose, no matter C. hutchinsonii growing on glucose or cellulose. There are 89 and 84 enriched membrane proteins growing on glucose and cellulose, respectively, which might be related to cellulose adherence. No matter cultivated on glucose or cellulose, forty of all these detected membrane proteins were both enriched when binding to cellulose. Furthermore, seventeen membrane proteins perhaps related to cellulose degradation or adherence were identified. Nine of them were the same as the proteins predicted by Gary Xie, et al. in 2007. The other eight membrane proteins r were not reported before. Based on their predicted functions and Itraq results, three categories were classified:two of them are only related to cellulose adherence; seven of them are only related to cellulose degradation; eight of them are related to cellulose adherence and cellulose degradation. Future studies are needed.

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