| Myxobacteria are a group of gram-negative bacteria with social behavior. Myxobacteria harbor great genome ranging from5Mbp to14.78Mbp endowing them ability to adapt wide-range environment. Myxobacteria are another group of secondary metabolites producer after Actinomyces, Cyanobacteria, and filamentous fungi. The genes encoding secondary metabolites enzymes account for5%of the genome size of sequenced myxobacteria. More than500different basic compounds and approximately1500structural variants have been characterized, but great potentials wait for exploring. The yields of secondary metabolites are very low in native host, instead of former fermentation optimization; the strategies like heterologous expression and combinatory biology have been applied to improving the yields of secondary metabolite as the development of synthetic biology. However, the active protocol of genetic manipulation is rare in myxobacteria. In model organism Myxococcus xanthus DK1622, genome edit conducts through double-time homologous recombination, while in Sorangium cellulosum, gene inactive conducts through suicide plasmid. Both of methods have long experimental periods.Since the launch of sequencing project of M. xanthus DK1622by Monsanto Company,17genome projects have registered in NCBI database and15genomes, either full sequence or draft genome are available. Comparatively, the researches in myxobaterial genome are few and far between. In this thesis, we comprehensively applied the genome, transcriptome and high throughput sequencing of PCR product based on Next Generation Sequencing technology to research the genomic structure, expression mode, as well as the core gene set of multiple myxobacterial strains. On the basis of genomic and transcriptomic analysis, we improved the protocol of conjugation in S. cellulosum, as well as, applied CRISPR/Cas9technology in M. xanthus DK1622to conduct genome editing and transcriptional interference.Complex environmental conditions can significantly affect bacterial genome size by unknown mechanisms. The So0157-2strain of S. cellulosum is an alkaline-adaptive epothilone producer that grows across a wide pH range. Here, we show that the genome of this strain is14,782,125base pairs,1,75-megabases larger than the largest bacterial genome from S. cellulosun reported previously. The total11,599coding sequences (CDSs) include massive duplications and horizontally transferred genes, regulated by lots of protein kinases, sigma factors and related transcriptional regulation co-factors, providing the So0157-2strain abundant resources and flexibility for ecological adaptation. The comparative transcriptomics approach, which detected90.7%of the total CDSs, not only demonstrates complex expression patterns under varying environmental conditions but also suggests an alkaline-improved pathway of the insertion and duplication, which has been genetically testified, in this strain. These results provide insights into and a paradigm for how environmental conditions can affect bacterial genome expansion.In alkaline condition, the expression of genes encoding CRISPR/Cas system and restriction and modification system significantly decreased in S. cellulosum So0157-2. Hereby, we compared the efficiency of conjugation of So0157-2in pH7.0and pH9.0conditions. The results showed the efficiency of conjugation in pH9.0was10times higher than that in pH7.0, which proved that the So0157-2strain tended to accept exogenous genetic material in alkaline condition and may contribute to the genome expansion. In addition, we identified a series of mutates with insertion in the genes encoding modification enzyme of epothilones, which prepared a pool to screen and research the variants of epothilones.Isolation by environment and/or distance is often suspicious and contentious in microorganisms for their ’unlimited’ dispersal and gene flow capabilities. S. cellulosum is a non-predatory social myxobacterial species. Here we show that ten tested S. cellulosum strains markedly varied in genome size, ranging from9.81Mbp to14.78Mbp. The core genome of the ten S. cellulsoum was only3.37Mbp, accouting for22.8%of So0157-2genome and34.4%of So0157-25genome. Comparing with the ratio of core genome of main bacterial and archaeal groups in species and genera level, the core genome ratio of S. cellulosum lay in the genera level. The ClonalFrame and GenoPlast analyses indicated mutation drove divergence of the small core genome, resulting in phylogenetic separation in S. cellulosum, while duplication and horizontal gene transfer were the major driving force to develop the varied big accessory genomes, suggesting for environment adaptation. Genome differentiation in S. cellulosum reflected the geographical separation but with exceptions. The sympatric genome variation and genealogy misplacement suggested that, in addition to the synergistic functions of environment and distance, intraspecies interactions played an important role in the population differentiation in S. cellulosum. The result puts new insights into intraspecies population differentiation and genome evolution in microorganisms.Myxococcus is another main group of myxobacteria besides Sorangium. Microorganisms rely on their alternative transcriptomic profiles to adapt to environmental fluctuation. When salt-tolerant Myxococcus cells are moved to a seawater environment, they change their growth, morphology, and developmental behavior. Comparative genomics analysis show the orthologous genes experienced strongly purifying selection in M. fulvus and M. xanthus after the long-term adaptation to the marine environment. Further comparative transcriptomics analysis showed different expression mode in various salt concentration. Genes encoding systems for energy production and ribosomal synthesis tend to keep silent in20%salt concentration, which the cells grew best. More genes were expressed or regulated under osmotic pressure and showed0%and50%salt concentration was a stressful environments for HW-1strain. Non-coding RNA was systematically described in M. fulvus HW-1and some evidences indicated that small RNA and anti-sense RNA involved in the regulation of salt adaptation. Here, we proposed the putative life style conversion of M. fulvus HW-1both in phenotype and molecular pathway.Comparative genomic analyses showed the core genome of myxobacteria was small and the expression of most non-essential genes was low. It suggested that most of these non-essential genes could be removed from genome in certain nutritional condition. For myxobacteria, the research of secondary metabolite needed a host with reduced genome. In order to identify the core and essential gene set, we created a random mutants pool of M. xanthus DK1622using pminiHimar-lacZ plasmid with an MmeI site in the invert repeat region. The genomic was digested using Mmel and an adaptor was added to nicked end. A450bp PCR product library was constructed to sequence using Illumina Miseq3000. The sequencing results were mapped to the genome of DK1622and identified the essential genes in CTT cultural media were345.In M. xanthus DK1622, the double-time homologous recombination protocol can knock out large to100kbp fragment in genome. However, the time to get purified mutant was longer than two months, so this method is not suitable to consecutive genome reduction. The recent rising CRISPR/Cas9technology have advance in efficient genome editing, but the related system is still absent from M. xanthus DK1622. In this study, we integrated the pMFl replication origin, the chloramphenicol resistant gene with aphll promoter, the (d)cas9gene with groEL promoter, and p15A origin into a plasmid called pCas9myxo. In the presence of appropriate CRISPR DNA sequences, this system could efficiently knock out target sequence and just need one round antibiotic screening, as well as, this system also can interfere the expression of target when using deficient dcas9gene.In summary, we illuminated the genomic structures and transcriptomic mode of S. cellulosum and M. fulvus using hybrid omic strategies, as well as, unveiled the population structure and differentiation of S. cellulosum in genome level. By hybrid using of random mutation and Tn-seq, we identified the core gene set of M. xanthus DK1622in nutritional condition. Finally, we successfully introduced the CRISPR/Cas9technology into the research of M. xanthus DK1622. |
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