FLP-FRT-Based Method To Obtain Unmarked Deletions Of Cytophaga Hutchinsonii And Preliminary Study Of Genes Related To The Type Ⅸ Secretion System

Energy, resources and the environment have been severe challenges faced by human existence and development since the21st century. Biomass resources are fixed solar energy, and plant biomass is the most abundant sustainable energy sources available to humanity in the globe. Bioconversion of lignocellulose which is the main component of plant biomass has been valued by many countries around the world. Lignocellulose consists of cellulose, hemicellulose and lignin, which comprises approximately35to50%,20to35%and5to30%of plant dry weight, respectively. An important feature of cellulose is its crystalline structure, which is hard to be digested by enzymes. The conversion of cellulose especially crystalline cellulose has been one of the bottlenecks in the lignocellulosic ethanol industry. Therefore, improvement of the converting efficiency of crystalline cellulose is the key to the optimum lignocellulose utilization.Cytophaga hutchinsonii is a widely distributed Gram-negative cellulolytic bacterium which belongs to the phylum Bacteroidetes. C. hutchinsonii can digest crystalline cellulose rapidly and thoroughly, and exhibits gliding motility over surfaces. Cellulose degradation by C. hutchinsonii needs direct contact with cellulose, and most of the cellulase activity seems to be cell associated. The analysis of C. hutchinsonii genome shows it doesn’t encode any proteins containing dockerin or cohesion domains which are the characteristics of cellulosomes. Thus, the mechanism of cellulose degradation by C. hutchinsonii is novel and different from the well-known strategies applied by many aerobic microorganisms and anaerobic microorganisms, which are the free cellulase mechanism and the multienzyme cellulosomes respectively. The study of the novel cellulose degradation strategy by C. hutchinsonii helps to reveal the mystery of efficient crystalline cellulose utilization, and serves the lignocellulose bioconversion industry in future. Moreover, a novel protein secretion system has recently been discovered in Porphyromonas gingivalis and Flavobacterium johnsoniae of the phylum Bacteroidetes, named as type IX secretion system （T9SS）. T9SS is linked to Bacteroidetes gliding motility and pathogenesis. The study of the T9SS in C. hutchinsonii may help to reveal the mystery of gliding motility.Due to the lack of genetic manipulation tools, the studies on mechanisms of cellulose degradation and gliding motility by C. hutchinsonii have remained stagnant until the transposon-mediated mutagenesis by conjugation was developed. Recently, some progress has been made in these two areas owing to the development of many other genetic manipulation tools. In this study, a method based on the FLP-FRT recombination system and linear DNA double crossover recombination was developed for C. hutchinsonii to generate unmarked deletions of both single genes and large genomic fragments. Using this method, we deleted dozens of genes that might be involved in cellulose degradation and gliding, including the components of the T9SS, and several functional genes were identified and studied. Comparative transcriptome analysis of C. hutchinsonii in media containing glucose versus crystalline cellulose as sole carbon sources was also discussed.1. Development of unmarked deletion method based on the FLP-FRT recombination system and linear DNA double crossover recombinationIn order to target genes independent of plasm id transformation, we tried to introduce linear DNA into C. hutchinsonii as done in other species. We constructed template plasmids with resistance genes flanked by multiple cloning sites for inserting homologous arms. The template plasmids were named pSJHS and pSJHC with ermF and cat as the resistance genes, respectively. After trying different fragment sizes and linear DNA concentrations, we found that～1μg linear PCR product with at least1.5kb of homologous sequence for each arm transformed to approximately109electrocompetent cells was adequate for obtaining transformants.The FLP-FRT recombination system from Saccharomyces cerevisiae is one of the most useful tools for efficient genetic engineering, and has proven functional in diverse bacterial species. Because the transcription elements in the phylum Bacteroidetes are different from those in Proteobacteria, this recombination system has not yet been used in Bacteroidetes. To apply the FLP-FRT recombination system to unmarked mutagenesis, several plasmids were constructed to customize the system for C. hutchinsonii, including two erythromycin-resistant template plasmids, two cefoxitin-resistant template plasmids and a FLP recombinase helper plasmind. Target gene was replaced by linear DNA double crossover recombination, followed by transformation of the FLP recombinase helper plasmid to evict the resistance gene. The FLP recombinase helper plasmid could be easily cured from non-selective culture. We obtained several unmarked deletions using this method, indicating it is an efficient method for unmarked deletion in C. hutchinsonii.One of the unmarked deletions was an unannotated gene, CHU₁165. The deletion of CHU₁165resulted in defects in cellulose degradation and motility on both wet agar and glass surfaces. Complementation of the mutant restored all the abilities, indicating that CHU₁165played an important role in cellulose degradation and motility. Bioinformatics analysis indicated that CHU₁165might encode a thiol-disulfide oxidoreductase, and further study needs to reveal its function in C. hutchinsonii.2. Implementation of the FLP-FRT-based method for deletion of large genomic fragments in C. hutchinsoniiThe identification of unannotated genes depended mainly on transposon-mediated mutagenesis. However, the insertion of transposons was not completely random, and the insertion biases made it difficult to construct a genome-wide mutant library. More than forty percent of C. hutchinsonii genes are unannotated, and identification of functions of these genes would consume a lot of time and effort. Using our method, specific large fragments of the C. hutchinsonii genome could be targeted. In this study, we obtained four mutants of large fragment deletions only by three or four times transformation, and the size of the excised fragment varied from9to19kb, spanning from6to22genes. Our method not only helps to identify unannotated genes more efficiently, but also may be a useful tool for identifying conditionally essential genes of C. hutchinsonii.One of the targeted large fragments was the region from CHU₃190to CHU₃202which contains four genes annotated as gspD, E, G and F. These four genes belong to the type Ⅱ secretion system （T2SS）. These are the only annotated genes related to those of the defined secretion systems across the outer membrane in C. hutchinsonii besides T9SS. Deletion of this putative T2SS operon showed no defects in cell growth, filter paper degradation and motility, suggesting these genes were not essential for cellulose degradation and motility of C. hutchinsonii.3. Preliminary study of genes related to the type IX secretion system in C. hutchinsoniiRecent studies have shown that some outer membrane proteins may play an important role in cellulose degradation by C. hutchinsonii with a finding supported by the isolation of an endoglucanase ChCel5A from the outer membrane. Thus, protein secretion system across the outer membrane may play a role in cellulose degradation. Analysis of the C. hutchinsonii genome showed a full set of orthologs of the T9SS. However, functions and physiological roles of the T9SS in C. hutchinsonii remain unknown. We targeted several genes of the T9SS by our method, and two genes （CHU₃237and CHU₀170） were identified to be involved in the cellulose degradation and motility.Unmarked deletion of CHU₃237（porU）, an ortholog of the C-terminal signal peptidase from P. gingivalis caused defects in colony spreading on agar, gliding on glass and cellulose degradation, but not the arrangement of cells on cellulose fiber. The results of SDS-PAGE and LC-MS/MS analysis of extracellular proteins showed that dozens of extracellular proteins were absent from the culture fluid of the A3237mutant, and many of them were putative substrates of the C. hutchinsonii T9SS. Western blot analysis of CHU₀344, which is one of the putative substrates of the T9SS confirmed that CHU₃237appeared to function as a CTD peptidase in C. hutchinsonii. Disruption of CHU₀170, an ortholog of porP resulted in defect in cellulose degradation, especially crystalline cellulose. The mutant also showed partial defects in gliding motility on glass and colony spreading on soft agar, but no defect in colony spreading on hard agar. CHU0170was predicted to be a membrane protein, and the loss of it caused decreased levels of some of the outer membrane proteins and extracellular proteins compared to the wild type. Further study needs to elucidate the function of CHU0170in C. hutchinsonii.In addition, another two components of the T9SS, CHU₂610and CHU₂225were identified as redundant or semi-redundant genes in C. hutchinsonii. The deletion of them showed no defects in cellulose degradation. CHU₀029, the ortholog of sprA could not be deleted completely, and the incomplete deletion mutant showed severely decreased viability. The other core genes of the T9SS could not be deleted either, indicating that the T9SS played important roles in not only cellulose utilization, motility and protein secretion but also some other aspects different from F. johnsoniae and P. gmgivalis.Our work supported that the T9SS existed in C. hutchinsonii and played important roles in cellulose utilization, motility and protein secretion. The core genes of the T9SS might be conditionally essential genes of C. hutchinsonii. Further study needs to focus on the mechanism how the T9SS functions in cellulose degradation and motility. The number of the putative T9SS substrates was more than a hundred, and the relationship between these substrates and cellulose degradation needs to be elucidated. CHU₀834-0841show high sequence identities to each other. These proteins are putative substrates of the T9SS except for CHU₀839. As described above that the T9SS plays an important role in cellulose degradation by C. hutchinsonii, the study of the T9SS substrates is important. Deletion of the fragment from CHU₀834to CHU₀841and preliminary results showed that these putative substrates of the T9SS were dispensable for cellulose utilization and motility.4. Comparative transcriptome analysis of C. hutchinsonii and deletions of some of the differentially expressed genes RNA sequencing was used to analyze the transcriptomes of C. hutchinsonii in media containing glucose versus crystalline cellulose as sole carbon sources, and more than a thousand differentially expressed genes were discovered. The trancriptome analysis showed that four candidate endoglucanases could be divided into two groups, among which CHU₂103and CHU₁280had relatively high levels of background transcription, while CHU₁107and CHU₁655responded more severely to the induction of crystalline cellulose. This reveals the exquisite regulatory mechanism of cellulases in C. hutchinsonii. However, most of the differentially expressed genes were unannotated and could not be subjected to KEGG pathway enrichment analysis.Dozens of differentially expressed genes were selected to be targeted. A dozen of regulators and histidine kinases were disrupted, but no mutants showed obvious defect in filter paper degradation. The deletions of the other genes encoding up-regulated cellulases and differentially expressed hypothetical protein showed similar results. The regulation of gene expression in C. hutchinsonii may be complicated and exquisite. Thus, the study of the regulation mechanism of cellulose utilization in C. hutchinsonii needs substantial efforts and time.In this study, the method based on the FLP-FRT recombination system and linear DNA double crossover recombination has been used to generate deletions of both single genes and large genomic fragments. Our method provides an efficient tool for quickly screening unannotated genes or gene clusters, which may promote the study on the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.