Metagenomic Studies On Cyanobacterial Blooms In Lake Taihu

The massive development of bloom-forming cyanobacteria is causing serious problems in eutrophic water bodies worldwide.Many cyanobacterial species have been reported to produce toxins,which threaten aquatic ecosystems and cause serious and occasionally fatal human liver,digestive,neurological,and skin diseases.Despite extensive researches in hydrometeorology,food chains,and genomics,the mechanism underlying the cyanobacterial bloom formation remains largely elusive.Microbial interactions with other microbes and their environment play a significant role in maintaining equilibrium among the various components of biological ecosystems.In cyanobacterial blooms,various microorganisms reportedly interact with bloom-forming cyanobacterial species.For understanding the mechanisms of cyanobacterial bloom formation,exploring the interactions among the bloom-forming cyanobacterial species and other co-existing species is indispensable.By integrating metagenomics,nutrient hydrology,meteorology and environmental factor datasets,the present study reveals the mechanism of cyanobacterial bloom formation from the perspective of bacterial diversity and metabolic pathways in Lake Taihu,a freshwater lake located in the Yangtze Delta Plain,Wuxi,China.The main findings of the study are summarized below.(1)An experimental platform method based on the 16S rRNA amplicon was established for a bacterial diversity survey of Taihu Lake.Reliable extraction of DNA is a key step in identifying the bacterial diversity and community composition of environmental samples.To find a reliable DNA extraction method,this study evaluated four widely used DNA extraction methods-CTAB,XS,the commercial Mobio DNA solation kit and the Omega DNA solation kit-on three kinds of samples collected from Lake Taihu.For comprehensive assessments,various factors(yield,purity,and integrity of Meta DNA)were considered along with the“spectrum”metric(a performance measure of the 16s rRNA V regions).XS was found to be most suitable for cyanobacterial aggregates(attached bacteria),whereas the Omega kit best analyzed the original water(total bacteria)and filtered water(bacterioplankton).After evaluating the performances of the V regions by the metrics“coverage”,“specificity”,"spectrum",and "POAOs",in silico and validation experiments with respect to SILVA SSU Ref 123NR,the V4 region achieved the best domain specificity,higher coverage and broader spectrum coverage in the bacterial domain.S-D-Bact-0564-a-S-15/S-D-Bact-0785-b-A-18 was determined as a promising primer set for surveying the bacterial diversity in eutrophic lakes.Meanwhile,Greengenes more robustly aligned the sequences reads recovered from Lake Taihu than the SILVA,RDP and Freshwater reference datasets.(2)The bacterial succession pattern and dynamics in the community structures in Lake Taihu were then observed.Cyanobacteria,Proteobacteria,Verrucomicrobia,Actinobacteria,and Planctomycetes were the dominant bacterial groups across all four seasons in Lake Taihu.However,the community structure significantly differed among the four seasons,revealing an obvious seasonal succession pattern of bacteria in the lake.The succession between any two seasons was significantly different,and the main successional groups were Microcystis,ACK-Ml,Clll,Synechococcus,Actinomycetales,Pirellulaceae and Sphingobacteriaceae.More specifically,Candidatus_Xiphinematobacter,Synechococcus and Flavobacterium were involved in the winter-spring succession,Microcystis,Synechococcus and Planctomyces in the spring-summer succession,Microcystis,Planctomyces and Opitutus in the summer-autumn succession,and Microcystis,Methylotenera,and Candidatus_Xiphinematobacte in the autumn-winter succession.The main drivers of the bacterial community succession were phytoplankton,water temperature,and N concentration.(3)The community diversity of BACA(Bacteria Associated with Cyanobacterial Aggregates,BACA)was ascertained,and the BACA structure was found to strongly depend on temperature.Bacterial community succession in Lake Taihu occurred on a short-term(weekly)basis as well as the long-term(seasonal)basis discussed above.The BACA of M.wesenbergii,M.panniformis,M.aeruginosa,and M.flosaquea revealed that different Microcystis host specific bacteria.Importantly,BACA was found to be involved in the carbon cycle and degradation of microcystin,nitrogen,and phosphorus,which affect the functions of their hosts.The above findings confirm an active role for BACA in cyanobacterial blooming.Indeed,BACA may more severely affect cyanobacterial aggregation than phytoplankton.Therefore,by exploring the interaction mechanism between cyanobacterial aggregates and bacteria,we can hope to unravel the mechanisms of cyanobacterial bloom formation from functional genes and metabolic pathways.(4)The complete genome of M.panniformis FACHB 1757 was sequenced and characterized.This Microcystis strain was isolated from Meiliang Bay of Lake Taihu in August 2011.The whole genome was sequenced using Pacific Bio RSII sequencer with 48-fold coverage.The complete genome sequence with no gaps contained a 5,686,839 bp chromosome and a 38,683 bp plasmid,encoding 6,519 and 49 proteins,respectively.Comparison with strains of M.aeruginosa and some other water bloom-forming cyanobacterial species revealed large-scale structure rearrangement and length variation at the genome level along with 36 genomic islands annotated genome-wide,which demonstrates high plasticity of the M.panniformis FACHB 1757 genome and reveals that Microcystis has a flexible genome evolution.Such highly plastic,flexibile genome evolution might explain the superior ecological competition and global distribution of Microcystis.(5)The MMCA(Meta-microbes of Cyanobacterial Aggregates,MMCA)diversity and metabolic pathways significantly differed among the different phases of cyanobacterial blooms.Those phases of cyanobacterial blooms were characterized by MBCA(Meta-bacteria of Cyanobacterial Aggregates,MBCA).The early blooming phase(January to March)was characterized by low abundance of Rheinheimera,Pseudomonas,and Phormidium.The exponential phase of cyanobacterial blooming(April to June)was dominated by Rheinheimera,Pseudomonas and Paucibacter,whereas the stationary phase(July to October)was rich in Microcystis,Bdellovibrio,Bryobacter,and Rickettsia.DNRA(Dissimilatory nitrate reduction to ammonium,DNRA),ANRA(Assimilatory nitrate reduction to ammonium,ANRA),and denitrification were the main processes occurring on MMCA for nitrogen metabolic pathway,and the nitrogen fixation proportion was comparatively low,with no detectable nitrification and anammox.DNRA and ANRA were observed in all three phases and particularly dominated in the N metabolic pathway,whereas no denitrification during the exponential and stationary phase of cyanobacterial blooms was observed.Notably,the P metabolic pathway did not significantly change throughout the three phases of cyanobacterial blooming.(6)Finally,this study revealed the mechanism of Microcystis and Dolichospermum succession in Lake Taihu.Host specificity was observed in both cyanobacterial species:Gemmatimonas was the dominant species in Microcystis,whereas Sediminibacterium and Saprospira were highly abundant in Dolichospermum.Meanwhile,DNRA,ANRA,and nitrogen fixation were observed in both Microcystis-and Dolichospermum-dominated blooms,but denitrification was absent in Microcystis blooms.Although Microcystis species are non-N2 fixing cyanobacteria,MMCA is a potentially functional unit for nitrogen fixation.This might explain the persistent dominance of the non-N2 fixing genus(Microcystis)in Lake Taihu.Addkionally,the continuously declining N load in Taihu Lake probably explains why Dolichospermum blooms dominate during winter and spring.The analysis further suggested that N and water temperature mainly drive the succession of Microcystis and Dolichospermum in Lake Taihu during those seasons.