| Although the discovery and recognition of more and more new marine microorganisms has been spurred on by the ever-growing progress of high throughput sequencing technology,there are a large number of uncultured groups among them.Therefore,it is of great significance to obtain a large number of strains of uncultured groups to study the physiology and biochemistry of marine microorganism and their contributions to marine ecosystems.Kiritimatiellota is found in a variety of habitats,and the uncultured taxa WCHB141 in this taxon is mainly distributed in the gastrointestinal tract of animals.Kiritimatiellota is a difficult-to-culture taxa,and only four cultureable strains have been published to date.Therefore,the culturable strains of the Kiritimatiellota taxa are urgently waiting to be discovered.Genomic analysis revealed that the Kiritimatiellota genome encodes a large number of carbohydrate active enzyme metabolic pathways,but lacks further phenotypic experimental data,which cannot characterize the preference of the Kiritimatiellota bacterial community for carbon sources,limiting its further enrichment and isolation culture.Based on 16S rRNA amplification and macrogenome sequencing techniques,this project focuses on the response of microbial communities to different macromolecular substrates,conducting analysis of differences in the structure and function of microbial communities,as well as isolation and cultivation of marine difficult to cultivate microorganisms,the Kiritimatiellota taxa.This article provides a research idea for studying the structure and function of microbial communities enriched by different macromolecular substrates,and also provides a solid research foundation for subsequent research on microbial ecology of the Kiritimatiellota taxa.The main research results are as follows:1.The analysis of the changes of bacterial community structure enriched by different substrates(carrageenan,fucoidan,tryptone,sodium alginate,Tween 80)by 16S rRNA amplicon sequencing showed that tryptone and carrageenan-enriched samples had the highest diversity of microbial communities,and Tween 80 samples had the lowest diversity.According to the enrichment time,the community diversity was highest on day 0 and the lowest on day 5.Different substrate samples were enriched with more Proteobacteria,Bacteroidota and Firmicutes.Fucoidan samples were enriched with more Kiritimatiellota,especially on day 5,the highest relative abundance in the samples reached 9.38%.2.In the isolation of culturable strains,a total of 360 bacterial strains were isolated by aerobic and anaerobic culture methods.The isolated strains were distributed in 5 phyla:Proteobacteria,Bacteroidota,Firmicutes,Verrucomicrobiota and Kiritimatiellota.A total of 6 potential novel species of Kiritimatiellota were isolated by the modified fucoidan medium,two of which belonged to the uncultured WCHB1-41 taxa,with the highest similarity of 83.12%,which is a potential novel class of Kiritimatiellota.3.Metagenomic sequencing of fucoidan substrate enrichment samples on days 5 and 12 found that both samples were annotated with more α-L-fucosidase and sulfatase,and the abundances detected in the enriched samples on day 5 were 1.91 times and 1.80 times higher than those in the samples on day 12,respectively.In addition,17 mediumhigh quality MAGs obtained by assembly,7 of which are MAGs in the Kiritimatiellota phylum.A comparative analysis of the genome of the phylum Kiritimatiellota showed that most of the culturable strains encoded a large number of fucoidan-related enzymes and nitrogenases,and most of the Kiritimatiellota phylum strains could undergo arsenate reduction process.None of the currently obtained genomes have been annotated with thiosulfate reductase,DMSO metabolism-related enzymes,and nitrate reduction-related enzymes,and most bacteria have the potential to use nitrite and arsenate as terminal electron acceptors and have the potential to produce acetate through fermentation.4.Complete the polyphasic classification of five Kiritimatiellota strains.Through comparative analysis of phenotypic characteristics,physiological and biochemical analysis and other methods,the taxonomic status of five bacterial strains such as strain B1221 was determined.A potential novel class and two potential novel genera of the Kiritimatiellota phylum have been established. |
