| Microorganisms are rich in diversity in nature.At present,the vast majority of global microorganisms are still uncultured,especially in the marine microorganisms.While the 'unculturable' majority of the bacterial world is accessible with cultureindependent metagenomics,etc.,the inability to study these bacteria using culturedependent approaches has severely limited our understanding of their physiological characteristics,ecological roles,and natural active products.Therefore,obtaining purely cultured strains of microorganisms that have not yet been cultured has important scientific significance and far-reaching application prospects.One of the key limiting factors that make it difficult for most microorganisms in nature to be purely cultivated is that there are symbiosis or interactions between different microorganisms in the original habitat,but it is difficult to simulate the interaction between microbial cells under laboratory conditions.Symbiosis and various metabolites and their intermediates,signal molecules,small molecule compounds and enzymes released by cells in the process of interaction.Microbial cultivation methods based on co-cultivation strategies are considered to be an effective method to overcome this problem.However,there are currently few studies on co-cultivation methods,and the existing methods still have many shortcomings,such as high contingency,inability to screen large quantities,complicated operations,and difficult purification.Therefore,it is urgent to develop a new type of co-cultivation technology to overcome the obstacles of microbial cultivation.This research carried out a systematic study on this issue,and achieved the following results:1.Based on commensalism of microbial symbiosis,this research designed a new type of co-cultivation technology—sandwich agar plate method,which is simple,efficient,easy to operate and can separate a large number of microorganisms.Based on the important ecological functions of rare species in microbial communities,we selects rare microbial species that can be cultured in the ecological environment as 'helper' strains to co-culture other marine microorganisms.In the study,the cell-active cultivable 'helper' strain was added and fixed in the middle layer of the double-layer medium,and the co-cultured microorganisms were isolated and cultured on the upper layer of the medium.The composite medium not only has the advantages of intuitive,simple,easy-to-operate and low technical difficulty of the traditional separation method,but also realizes physical isolation of the 'helper' and the target isolate and the technical difficulty of the purification of the target strain.2.Eight marine bacteria were selected as 'helper' species in the sandwich agar plate technology,and the technology was applied to the isolation of bacterial resources in Weihai offshore sediments.Experimental verification shows that the design of the sandwich agar plate technology is successful and effective for the separation and cultivation of uncultured microorganisms.In order to verify the effectiveness of the sandwich agar plate technology in the separation and culture of uncultured marine microorganisms,we selected 8 species of rare species in marine habitats as the 'helper' species,including Algibacter pectinivorans,Formosa spongicola,Salipiger mucosus,Cribrihabitans marinus,Roseibacterium beibuensis,Colwellia aestuarii,Shewanella xiamenensis and Marinobacter algicola,to apply the sandwich agar plate technology to the isolation of bacterial resources in the offshore sediments of Weihai.A total of 825 species were isolated in the experiment.Compared with the control medium without 'helper' the bacterial diversity in 8 types of the sandwich agar plate was 5 times;Among them,there are 383 potential new species,which is 8.5 times that of the control group,which shows that the sandwich agar plate method has great potential in the separation and culture of uncultured microorganisms.In addition,we used 16S rRNA gene amplicon sequencing technology to analyze the cultured bacterial community in different sandwich agar plates and the control medium.The results show that the addition of the 'helper' strains had a great impact on the cultured bacterial community,and different sandwich agar plates have their own unique bacteria community composition.3.317 novel strains isolated by interaction were co-cultured with'helper'strians.The results showed that the 'helper' strians had a real and effective effect on the isolated co-cultured strains.In order to explore the authenticity and effectiveness of the 'helper' on the growth of the isolated strains during the cocultivation process,we conducted co-cultivation experiments on 317 novel strains isolated by interaction and the 'helper' respectively.The results show that more than 50%of the strains can be promoted by 'helper'.These bacteria grows slowly or cannot grow under pure culture conditions,while co-cultivation with 'helper' can significantly promote them growth,showing that the use of sandwich agar plate technology can obtain pure culture of certain microorganisms that depend on symbiosis.4.The growth-promoting mechanism of some promoted strains in Bacillusceae and Flavobacteraceae were analyzed,and it was found that the small molecule compounds secreted by 'helper' strians can significantly promote the growth of dependent bacteria in Bacillusceae;The growth-promoting factor required by the 3 strains of Flavobatecriaceae is heme.By exploring the growthpromoting mechanism of some isolated strains in Bacillusceae and Flavobacteraceae,it was found that the small molecule compounds secreted by 'helper' strains Shewanella xiamenensis and Colwellia aestuarii can effectively promote the growth of dependent bacteria in Bacillusceae.At the same time,taking the growth-promoting phenomenon of 3 strains of Flavobacteraceae as an example,combined with cell culture and comparative genomics analysis,it was found that the growth-promoting factor was heme,and the interaction mechanism them and Colwellia aestuarii may be 'helper'provides them with heme-containing protein(mainly cytochrome).This is the first report that heme is an important growth promoting factor of uncultured microorganisms in a non-host environment.It also shows that heme not only plays a key role in the interaction between pathogenic microorganisms and host,but also plays an important role in the interaction between microbial cells in non host natural environment.5.The genomes of 113 promoted strains were analyzed,and the results showed that the important metabolite synthesis pathways in the promoted strains were missing to varying degrees.This may be a common feature of uncultured microorganisms;The analysis of molecular metabolites shows that most of these metabolites are essential substances for the growth of microorganisms.In order to fully understand and evaluate the important role of 'helper' in sandwich agar plates,we analyzed the genomes of 113 growth-promoting strains and the small molecule metabolites secreted by 3 'helper' strains.Genomic analysis results show that important metabolite synthesis pathways(such as fatty acid,nucleotide,amino acid,cofactor and vitamin synthesis,etc.)in the promoted strains are missing to varying degrees.It is speculated that the genome simplification expressed by the "Black Queen Hypothesis"may be an important factor restricting the pure culture of microorganisms.The addition of 'helper' may provide the necessary growth promoting factors with them;in addition,we used antiSMASH to predict the secondary metabolite gene clusters of the promoted strains.The results show that more than 96%of the strains may have multiple secondary metabolite gene clusters,which hints that the sandwich agar plate can be used not only for the excavation of microbial resources,but also for the discovery of new active substances.In the detection and analysis of the small molecule metabolites secreted by 3 'helper' strains,we found that most of these are required for the growth of microorganisms,such as known growth-promoting factors,and complex organic carbon and nitrogen sources,etc.The results show that 'helper' can provide a variety of usable substrates and growth factors for other microorganisms.It also fully demonstrates that the sandwich agar plate culture strategy is an effective co-cultivation method,which has broad application prospects in the future microbial resource mining. |
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