Biodiversity Study Of The Phycosphere Microbiota And Identification Of Novel Bacterial Species Isolated From Alexandrium Catenella

Harmful algal blooms?HABs?cause great harm to the marine environment and ecosystems,and seriously threaten human life and health through the food chain.The source of PSP is still inconclusive.It is actually produced by the algae itself,or by the phycosphere microbiota?PM?,or synergistically at the same time,which is still pending.The interaction between algae and bacteria is the key to the precise source of PSP,and the interpretation of the biodiversity information of its phycosphere microbiota is the necessary premise for the study of algae-bacteria interaction.Alexandrium catenella is the representative harmful algal blooms dinoflagellate worldwide.In order to clarify the species diversity information of phycosphere microbiota of A.catenell.In this study,two dinoflagellate A.catenella?AC 01 and AC 02?were used as materials.The 16S rRNA V3-V4 region and the full-length amplicon sequencing technology were used to compare and analyze the diversity of the PM.The species and abundance information in the samples were obtained,and the species differences of the PM of two A.catenella were compared.The results of 16S rRNA short read sequencing showed that the PM of AC 01 included 12 phyla,27 classes,59 orders,105 families and184 genera.The PM of AC 02 included 11 phyla,19 classes,52 orders,85 families and143 genera.The dominant phylum is Proteobacteria,accounting for 66.17%and 84.96%,respectively.The dominant genera are all Marivita,accounting for 14.4%and 26.4%of AC 01 and AC 02,respectively.The 16S rRNA full-length amplicon sequencing results showed that the AC 01 sample contained 4 phyla,8 classes,16 orders,14 families and 19genera,and the AC 02 contained 4 phyla.7 classes,13 orders,16 families and 22 genera.The dominant phylum are also Proteobacteria,accounting for 82.02%and 89.11%,respectively.The dominant genera are all Marivita,in AC 01 and AC 02 and 45.73%and38.72%.This genus is an important member of the Rhodobacteraceae.It is widespread in the marine environment and is closely related to the carbon and nitrogen cycles in the ocean.By comparing the results of the full-length 16S rRNA amplicon sequencing technology with the high-throughput sequencing,the proportion of new species exhibited by the full-length 16S rRNA amplicon sequencing has increased significantly,it realize the precise positioning of the taxonomic classification of the PM,thereby discovering the special novelty of PM of the A.catenella.Through pure culture technology,two culturable and potential new species were selectively separated,which were analyzed using polyphasic taxonomy and identified as new species Limnobacter alexandrii LZ-4 and Mameliella alexandrii Z6-9.Among them,L.alexandrii LZ-4 was gram-negative,rod-shaped,cell size is 0.4-0.6×1.1-1.8?m,light yellow,with flagella.The growth conditions are 10-42 ^oC?optimum 33^oC?,pH 5.5-9.0?optimum pH 7.0?and 0-7%?w/v?NaCl?optimum 3.5%?.Polar lipids include diphosphatidylglycerol?DPG?,phosphatidylethanolamine?PE?,phosphatidylglycerol?PG?,two unidentified amino lipids?ALs?and three unknown polar lipids?Ls?.The main respiratory quinone is Q-8.The main cell fatty acids are summed feature 8(C_18:1?7c and/or C_18:1?6c),summed feature 3(C_16:1?7c and/or C_16:1?6c),C_16:0,C_17:0 cyclo and C_10:0 3-OH.The G+C content is 52.5 mol%.M.alexandrii Z6-9 is gram-negative,non-exercise and aerobic.The cell size is 0.9-1.5?m x 2.1-3.4?m.The growth conditions are15-40 ^oC?optimum 25 ^oC?,pH 5-10?optimum pH 7.0?and 1-10%?w/v?NaCl?optimum3.5%?.The main respiratory quinone is Q-10.Polar lipids include phosphatidylglycerol?PG?,phosphatidylethanolamine?PE?,glycolipid?GL?,phosphatidylcholine?PC?,one unidentified phospholipid?PL?,and four unidentified lipids?Ls?.The main fatty acid components are Summed feature8(including C_18:1?7c and/or C_18:1?6c),C_18:1?7c 11-methyl,C_19:0?8c cyclo and C_18:0.The genomic DNA G+C content is 64.9 mol%.Genome sequencing was performed on two new species obtained by Illumina Hiseq4000.Among them,the total length of L.alexandrii LZ-4 genome is 3488419 bp,which contains 17 contigs,N₅₀ is 452966 bp,G+C%is 52.5%,and the number of annotated genes is 3298.The total length of M.alexandrii Z6-9 genome is 5663795 bp,which contains 38contigs,N₅₀ is 326544 bp,G+C%is 64.9%,and the number of annotated genes is 5543.Using the carbohydrate active enzyme database for annotation,the number of proteins related to the polysaccharidelyase in L.alexandrii LZ-4 and M.alexandrii Z6-9 were found to be 2 and 3,respectively,indicating that the ability to utilize polysaccharides is limited.Antismash software was used to predict and analyze metabolites.The results showed that strain M.alexandrii Z6-9 had the genetic basis for the synthesis of ectoine and carotenoid.