微小亚历山大藻amtk-4共附生菌群多样性及其产毒新种Z1-D的sxtA1基因进化研究
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摘要
藻菌关系是揭示赤潮生消与防控、赤潮毒素产生机制的关键,而产毒甲藻共附生菌群多样性及可培养菌株的获得是解析藻菌关系的前提。微小亚历山大藻(Alexandrium minutum)是全球性典型赤潮甲藻,其产生的麻痹性贝类毒素(PSP)危害巨大,但目前对其共附生菌群尚缺乏系统性研究。通过高通量测序首次解析了产毒微小亚历山大藻amtk-4共附生菌群的物种种类及相对丰度信息;分离获得可培养菌株并对筛选获得的产毒细菌新种Z1-D的毒素合成基因sxt A1进行了基因进化分析,结果表明,amtk-4共附生菌群包括85个OTU,其中包括10门、20纲、40目、59科及87属。其6个优势属包括Phycisphaeraceae科未知属(11. 8%)、Muricauda属(10. 3%)、腐螺旋菌科未鉴定属(9. 1%)、Hyphomonadaceae科未鉴定属(8. 9%)、Haliea属(5. 7%)及红细菌科未鉴定属(5. 1%)。amtk-4共附生菌群中未鉴定属比例高达53. 4%。藻生长稳定期所分离获得的可培养细菌数量及种类最多。5株细菌中菌株Z1-D及Z1-4经分子鉴定分别为亚硫酸杆菌属(Sulfitobacter)及Mesorhizobium属新种。其中Z1-D发酵代谢产物含微量石房蛤毒素(STX),其基因片段orf-01498与蓝藻sxt A1基因高度同源,与产毒蓝藻间可能存在着基因共同进化。
Algae-bacteria interaction study is essential for revealing mechanisms of the occurrence and prevention of harmful algal blooms( HABs),and paralytic shellfish poisoning( PSP) toxins biosynthesis. The discovery of the biodiversity of bacterial community is the primary prerequisite for algae-bacteria interaction study. Alexandrium minutum,a typical red-tide causing dinoflagellate with global distribution,produces PSP toxins with huge hazard. However,few achievements are available on the biodiversity of algae associated bacterial community at present time. The species types and their relative abundance of the bacterial community of A. minutum amtk-4 were analyzed using Illumina Miseqhigh-throughput sequencing.Meanwhile,the cultivable bacterial strains from amtk-4 were also isolated. The gene evolution analysis of sxtA1 gene of the novel species strain,Z1-D,was performed. Based on the results obtained,the bacterial community of amtk-4 included 10 phylum,20 classes,40 orders,59 families and 87 genera. Six dominant genera including unclassified genus of family Phycisphaeraceae( 11. 8%),Muricauda sp.( 10. 3%),unclassified genus of family Saprospiraceae( 9. 1%), unclassified genus of family Hyphomonadaceae( 8. 9%),Haliea sp.( 5. 7%) and unclassified genus of family Rhodobacteraceae( 5. 1%) were found.Totally 53. 4% of the bacterial flora belonged to the novel genus. Two cultivable bacterial strains isolated,named as Z1-4 and Z1-D,were identified as novel species of Sulfitobacter and Mesorhizobium,respectively.Furthermore,strain Z1-D can produce trace saxitoxin in the fermentation products,and the gene fragment of orf01498 in its genome showed high similarity with sxtA1 genes from cyanobacteria,indicating that strain Z1-D and cyanobacteria might have co-evolution relationship on toxic sxtA1 gene.
Algae-bacteria interaction study is essential for revealing mechanisms of the occurrence and prevention of harmful algal blooms( HABs),and paralytic shellfish poisoning( PSP) toxins biosynthesis. The discovery of the biodiversity of bacterial community is the primary prerequisite for algae-bacteria interaction study. Alexandrium minutum,a typical red-tide causing dinoflagellate with global distribution,produces PSP toxins with huge hazard. However,few achievements are available on the biodiversity of algae associated bacterial community at present time. The species types and their relative abundance of the bacterial community of A. minutum amtk-4 were analyzed using Illumina Miseqhigh-throughput sequencing.Meanwhile,the cultivable bacterial strains from amtk-4 were also isolated. The gene evolution analysis of sxtA1 gene of the novel species strain,Z1-D,was performed. Based on the results obtained,the bacterial community of amtk-4 included 10 phylum,20 classes,40 orders,59 families and 87 genera. Six dominant genera including unclassified genus of family Phycisphaeraceae( 11. 8%),Muricauda sp.( 10. 3%),unclassified genus of family Saprospiraceae( 9. 1%), unclassified genus of family Hyphomonadaceae( 8. 9%),Haliea sp.( 5. 7%) and unclassified genus of family Rhodobacteraceae( 5. 1%) were found.Totally 53. 4% of the bacterial flora belonged to the novel genus. Two cultivable bacterial strains isolated,named as Z1-4 and Z1-D,were identified as novel species of Sulfitobacter and Mesorhizobium,respectively.Furthermore,strain Z1-D can produce trace saxitoxin in the fermentation products,and the gene fragment of orf01498 in its genome showed high similarity with sxtA1 genes from cyanobacteria,indicating that strain Z1-D and cyanobacteria might have co-evolution relationship on toxic sxtA1 gene.
引文
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[2] KODAMA M. Paralytic shellfish poisoning toxins:Biochemistry and origin[J]. Aqua-Bioscience Monographs,2010,3(1):1-38.
[3] BERDALET E,FLEMING L E,GOWEN R,et al.Marine harmful algal blooms, human health and wellbeing:Challenges and opportunities in the 21st century[J]. Journal of the Marine Biological Association of the United Kingdom,2016,96(1):61-91.
[4] BACKER L C, MANASSARAM-BAPTISTE D,LEPRELL R, et al. Cyanobacteria and algae blooms:Review of health and environmental data from the harmful algal bloom-related illness surveillance system(HABISS)2007-2011[J].Toxins,2015,7(4):1048-1064.
[5] WANG D Z,ZHANG S F,ZHANG Y,et al. Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates:A molecular overview[J]. Journal of Proteomics,2016(135):132-140.
[6] MELLO D F,DA SILVA P M,BARRACCO M A,et al. Effects of the dinoflagellate Alexandrium minutum and its toxin(saxitoxin)on the functional activity and gene expression of Crassost reagigas hemocytes[J]. Harmful Algae,2013(26):45-51.
[7] ZHANG X L,MA L Y,TIAN X L,et al. Biodiversity study of intracellular bacteria closely associated with paralytic shellfish poisoning dinoflagellates Alexandrium tamarense and A. minutum[J].International Journal of Environment and Resource,2015(4):23-27.
[8] CHOU H N,CHEN Y M,CHEN C Y. Variety of PSP toxins in four culture strains of Alexandrium minutum collected from southern Taiwan[J].Toxicon,2004,43(3):337-340.
[9] YANG Q,ZHANG X L,LI L Z,et al. Ponticoccus alexandrii sp. nov.,a novel bacterium isolated from the marine toxigenic dinoflagellate Alexandrium minutum[J]. Antonie van Leeuwenhoek, 2018(111):995-1000.
[10] YANG Q,JIANG Z W,HUANG C H,et al. Hoeflea prorocentri sp. nov.,isolated from a culture of the marine dinoflagellate Prorocentrum mexicanum PM01[J]. Antonie van Leeuwenhoek,2018(111):1845–1853.
[11] ZHANG X L, TIAN X Q, MA L Y, et al.Biodiversity of the symbiotic bacteria associated with toxic marine dinoflagellate Alexandrium tamarense[J]. Journal of Biosciences and Medicines,2015,3(06):23-28.
[12] STUKEN A,RIOBO P,FRANCO J,et al. Paralytic shellfish toxin content is related to genomic sxt A4copy number in Alexandrium minutum strains[J].Frontiers in Microbiology,2015,6:404.
[13] DIA A,GUILLOU L,MAUGER S,et al. Spatiotemporal changes in the genetic diversity of harmful algal blooms caused by the toxic dinoflagellate Alexandrium minutum[J]. Molecular Ecology,2014,23(3):549-560.
[14] GREEN D H,LLEWELLYN L E,NEGRI A P,et al. Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum[J]. FEMS Microbiology Ecology,2004,47(3):345-357.
[15]唐莹莹,乔玉宝,蒋志伟,等.东海产麻痹性贝毒链状亚历山大藻共生菌群的物种多样性研究[J].海洋渔业,2018,40(6):721-727.TANG Y Y, QIAO Y B, JIANG Z W, et al.Biodiversity study of the bacterial community associated with toxic marine dinoflagellate,Alexandrium catenella LZ1706,from East China Sea[J]. Marine Fisheries,2018,2018,40(6):721-727.
[16] PARK A Y,TEERAVET S,PHENG S,et al.Sulfitobacter aestuarii sp. nov. a marine bacterium isolated from a tidal flat of the Yellow Sea.[J].International Journal of Systematic&Evolutionary Microbiology,2018,68(5):1771-1775.
[17] ZHANG J,GUO C,CHEN W,et al. Mesorhizobium wenxiniae sp. nov. isolated from chickpea(Cicer arietinum L.)in China.[J]. International Journal of Systematic&Evolutionary Microbiology,2018,68(6):1930-1936.
[18] AYRAPETYAN M,WILLIAMS T C,OLIVER J D.Bridging the gap between viable but non-culturable and antibiotic persistent bacteria[J]. Trends in Microbiology,2015,23(1):7-13.
[19] LANDRY Z C,VERGIN K,MANNENBACH C,et al. Optofluidic single-cell genome amplification of sub-micron bacteria in the ocean subsurface[J].Frontier in Microbiology,2018(9):1152.
[20] HOVER B M,KIM S H,KATZ M,et al. Cultureindependent discovery of the malacidins as calciumdependent antibiotics with activity against multidrugresistant gram-positive pathogens[J]. Nature Microbiology,2018(3):415–422.
[21] ORR R J S,STUKEN A,MURRAY S A,et al.Evolution and distribution of saxitoxin biosynthesis in dinoflagellates[J]. Marine Drugs,2013,11(8):2814-2828.
[22] SAVELA H,SPOOF L,HOYSNIEMI N,et al. First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes[J]. Advances in Oceanography and Limnology,2017,8(1):61-70.
[23] GALLACHER S,FLYNN K J,FRANCO J M,et al.Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp.(Dinophyta)in culture[J]. Applied and Environmental Microbiology,1997,63(1):239-245.
[24] GALLACHER S,SMITH E A. Bacteria and paralytic shellfish toxins[J]. Protist,1999,150(3):245-255.
[25] WANG S,DOS-SANTOS A L A,HUANG W,et al. Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria[J].Science,2017,357(6358):1399-1402.
[26] RAMANAN R,KIM B H,CHO D H,et al. Algaebacteria interactions:Evolution, ecology and emerging applications[J]. Biotechnology Advances,2016,34(1):14-29.
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