Mechanistic Insights Into Microbial Community Structure And Function In South China Sea And Union Glacier And Coexistence Of Copiotroph And Oligotroph In The Surface Seawater Of India Ocean

Oceans and polar regions are important ecosystems on earth.Oceans cover 71%of the earth's surface area.Physicochemical factors such as temperature,salinity,current and light affect dynamic changes of ocean.Polar regions are covered by snow all year round with extremely low temperature.It is particularly sensitive to climate impact.In the complex marine and polar environment,microorganisms are the foundation of food chain and play an important role in energy flow and matter circulation including carbon,nitrogen and sulfur cycle.Using metagenomic analysis,the study of microbial community change,geographical distribution,environmental response and ecological function is of great importance for elucidating the global chemical cycle of matter.Large-scale interactions between microbes can be explored through computer simulations,which could help verify an interaction through typical co-culture experiments.Many marine bacteria have evolved to grow optimally in copiotrophic or oligotrophic environements,allowing copiotroph and oligotroph to survive in different nutrient habitats.However,it's still unknown that how the copiotrophs and oligotrophs interact and coexist in the isotropic surface seawater environment when facing the same High-molecular weight(HMW)resource.In this dissertation,firstly,microbial community compositions,geographical distributions and environmental responses were studied in marine and polar ecological environments,and ecological functions of microorganisms were described.Secondly,Pseudoalteromonas sp.SW0106-04(P.sp.W04)and Brachybacterium sp.SW0106-09(B.sp.Y09)were taken as the research objects.Through physiological and genomic analyses,nutrition types and HMW substrate utilizations of the two strains were described.In addition,long-term co-culture studies help us to propose the co-existence model.Finally,through kinetic and comparative transcriptomic analyses,the coexistence mechanism of copiotrophs and oligotrophs in surface seawater was revealed.1.Vertical and horizontal biogeographic patterns and major factors affecting bacterial communities in the open South China SeaMicroorganisms display diverse biogeographic patterns in the three-dimensional contiguous seawater.The distance-decay relationship,the change in species composition similarity between different communities over a geographic distance,is a commonly observed biogeographic pattern.The ocean is a three-dimensional contiguous aquatic system where microbial diversity can be affected by horizontal and vertical distances.There are sinking and upwelling currents that can vertically mix seawater and microorganisms.However,it remains unclear if there are any differences between the patterns of distance-decay relationships along the vertical depth and horizontal distance,and if there are differences,it is unclear which factors drive these differences.To study biogeographic patterns and the corresponding driving forces,the bacterial distance-decay patterns along the horizontal and vertical dimensions in the South China Sea(SCS)were investigated through the sequencing of partial 16S rRNA gene regions.Along the horizontal geographical distances(up to?1000 km),no significant distance-decay pattern in community compositions was observed in any of the tested seawater layers.However,vertical depths(up to?4 km)had strong effects on bacterial community variation.Each stratification area produced homogeneous water masses harboring analogous physicochemical properties.Water mass differences at different pelagic depths appeared to form a dispersal barrier that led to the creation of different sets of taxa.In addition,RDA analyses showed that community variations in the vertical direction were strongly correlated with the prominent variation of environmental factors.Apparently,the changes in bacterial community compositions along vertical distances were much greater than those along horizontal distances.The results showed that the distance-decay relationship in bacterial communities at the medium spatial scale was associated with vertical depth rather than with horizontal distance,even though the horizontal distance is much larger than the vertical distance in the open SCS.2.Reconstruction of the functional ecosystem in Union Glacier,Antarctica through metagenomicsAntarctica is covered by multiple larger glaciers with diverse extreme conditions.Microorganisms in Antarctic regions are primarily responsible for diverse biogeochemical processes.Most previous studies of polar environments have focused on the less extreme sub-Antarctic and maritime Antarctic Peninsula locations,which have fundamentally different biomes.Aquatic microorganisms,for instance,have been found in diverse habitats including Antarctic lakes,ponds and coastal fringes.In the hyper-arid polar desert of the Dry Valleys,functional traits of specialized microbial communities from distinct lithic and soil niches have been identified.However,little is known about microbial communities from the high elevation glaciers such as the Union Glacier and almost nothing is known about the stress tolerance mechanisms for climatic extremes in edaphic Antarctic microorganisms.The Union Glacier,located in the inland of West Antarctica at 79°S,is a challenging environment for life to survive due to the high irradiance and low temperatures.Here,using metagenomic analyses,the functional microbial ecosystem adjacent to the Union Glacier was analyzed through the reconstruction of carbon,nitrogen and sulfur metabolic pathways.A low biomass but diverse microbial community was found.Although archaea were detected,bacteria were dominant.Taxa responsible for carbon fixation were comprised of photoautotrophs(Cyanobacteria)and chemoautotrophs(mainly Alphaproteobacterial clades:Bradyrhizobium,Sphingopyxis and Nitrobacter).The main nitrogen fixation taxa were Halothece(Cyanobacteria),Methyloversatilis and Leptothrix(Betaproteobacteria).These putative producers provide organic carbon and nitrogen for the growth of other heterotrophic microbes such as Dechloromonas(Betaproteobacteria)and Thermoleophilum(Actinobacteria).A range of metabolic pathways and genes related to high irradiance,low temperature and other stress adaptations were detected,which indicate that the microbial communities had adapted to and could survive in this hash environment.These results provide a detailed perspective of the microbial functional ecology of the Union Glacier area and improve our understanding of linkages between microbial communities and biogeochemical cycling in high Antarctic ecosystems.3.Bacterial community structure and profiles of enzyme-producing bacteria and their extracellular enzymes in the surface seawater of Indian OceanMonsoon-driven circulation could cause upwelling currents in the open ocean,with large-scale horizontal transportation of nutrition,especially in surface seawater.In addition,enough sunlight in the surface waters promotes photosynthesis and phytoplankton production.These active processes influenced horizontal microorganism production and movement without notable dispersal limitation.These microorganisms play critical roles in the mineralization of complex organic matters.HMW biopolymers are important components of organic matters of primary productions and these biopolymers must be hydrolyzed by extracellular enzymes to small molecules(<approximately 600 daltons)that can be absorbed and metabolized by microorganisms.Degradation of HMW biopolymers by heterotrophic bacteria initiated as an extracellular process.Therefore,it is necessary to study extracellular enzymes secreted by microorganisms.Firstly,we analyzed bacterial community structures at 41 sites in the surface waters of Indian Ocean using high-throughput sequencing method.Total 37 phyla were found.Proteobacteria dominated with the proportion of 76.06%,followed by Bacteroidetes,Cyanobacteria and Actinobacteria.The bacterial communities of central Indian Ocean were significantly different from those of southwest Indian Ocean.Secondly,cultivable bacterial strains were screened and totally 33 different strains were identified.These cultured strains belonged to four bacterial phyla.Proteobacteria was the predominant phylum,Vibrio and Pseudoalteromonas were the predominant genera.Extracellular enzymatic activities towards 8 HMW substrates on the plates were tested for 14 strains from 12 genera.The result showed that Sodium alginate was degraded by the most strains,followed by DNA.P.sp.W04 showed the highest degradation ability and could degrade DNA,casein and polysaccharides such as starch,chitin,which can be as a producer in the utilization of public goods exploitation.B.sp.Y09 only degraded one substrate,which can be as cheaters in microbial interaction.P.sp.W04 and B.sp.Y09 provides a foundation for studying interaction between different microorganisms.4.Experimental insights into nutritional types and the coexistence phenomenon of copiotroph P.sp.W04 and oligotroph B.sp.Y09 in the surface seawater of Indian OceanBacterial extracellular enzymes can be considered as a kind of public goods.Extracellular enzymes and enzymatic products can be used by the producers and surrounding cheaters that do not produce extracellular enzymes.Bacteria are never alone in nature.Considering the high bacterial species richness in most natural environments,such as soil and seawater,it can be anticipated that bacteria would interact and coexist not only with same species but also more frequently with different species.However,the study of bacterial cooperation and coexistence between two different species through public goods is rare.Generally,based on different trophic strategies,the marine bacteria could be classified into copiotroph and oligotroph categories.The genomes of copiotrophs encode more proportion of extracellular proteins including extracellular enzymes.Oligotrophs have less extracellular proteins and usually have no predicted extracellular enzyme.In continuous ocean system,when the enzyme-producing copiotrophs were present nearby,the oligotrophs can act as a cheater and utilize the hydrolysates from the degradation of HMW organic matters.However,it's still unknown that how the copiotrophs and oligotrophs interact and coexist in the isotropic surface seawater environment when facing the same HMW resource.Firstly,nutritional types of P.sp.W04 and B.sp.Y09 were analyzed.Strain W04 grew and decayed quickly in diluted mediums while strain Y09 can grow steadily and have higher final OD values.Y09 obviously grew better than W04 in diluted mediums that contained low concentration nutrients.In addition,strain W04 had relatively larger genome size,five 16S rRNA genes,higher proportions of extracytoplasmic proteins and two-component systems,many extracellular protease/peptidase,chitinase and amylase.In contrast,strain Y09 had genomic features of oligotrophic lifestyle:small genomic size,only one 16S rRNA gene,less or no extracellular enzymes and higher proportion of some oligotroph feature COG categories,such as COG0318 and COG1680.Thus,W04 had a copiotrophic lifestyle and Y09 had an oligotrophic lifestyleStrain W04 can grow in the casein and gelatin medium,showing W04 can degrade HMW proteinaceous substrates and utilize these substrates alone.Strain Y09 can not grow in the casein and fish gelatin medium,showing that Y09 can not utilize the HMW proteinaceous substrates by itself.Although Y09 can not utilize casein by itself,it could survive by using the enzymatic products of proteases secreting by other organisms.Besides,the transfer experiments showed that the W04 can quickly outcompete Y09 in the short coculture system.But long-term oligotrophic co-culture showed that the strain W04 and Y09 could form an oscillating coexistence equilibrium with the pulse input of nutrients.Finally,we present an oscillation coexistence model of copiotroph and oligotroph.5.Experimental insights into the coexistence mechanism of copiotroph P.sp.W04 and oligotroph B.sp.Y09 in the surface seawater of Indian OceanAccording to coexistence model of strain W04 and Y09,we further analyzed their coexistence mechanism.Hydrolysis of casein by protease can produce amino acids and peptides.We then tested Y09 using what substrates to grow.The result showed that Y09 could only utilize amino acids to grow.The amino acids utilization efficiencies were tested for strain W04 and Y09.The result showed that W04 can take up the amino acids thoroughly and quickly.At the 24th hour,strain W04 biomass can reach the peak and the amino acids in cultures were almost depleted.The growth rate of strain Y09 in amino acids medium was very slow.Its biomass reached the peak at the 10th day,and 75%of the added amino acids were still not used.This showed that W04 had much higher amino acids utilization efficiency than Y09.However,the adsorption rates test for cysteine showed that the cysteine adsorption rate of Y09 would larger than that of W04 when the cysteine concentration was less than 82 nM.Thus,the strain W04 can quickly outcompete Y09 in the coculture system under the condition of adequate nutrition.However,strain Y09 can grow better than W04 and have higher final OD values in low nutrient concentration.Transcriptomes of long-term coculture were profiled using RNA-seq approaches to investigate the gene expression variations of both strains.When new nutrients were added,genes of W04 related to tricarboxylic acid cycle,glycolysis,amino acid metabolism,ATP synthase,DNA/RNA and fatty acid biosynthesis pathways were up-regulated significantly.In addition,15 genes involved in secretion system,5 extracellular protease genes and 12 extracellular peptidase genes of W04 were also up-regulated.This showed that W04 could response quickly to extracellular casein addition and degrade it into amino acids and oligopeptides that were then used for growth.While there are much less genes that were up-regulated in Y09 for the nutrientil period and no clear gene regulation pattern was observed,showing that genes of Y09 were less regulated when nutrients were added.When nutrients were depleted,genes of W04 related to substrates degradation,energy production and biomass synthesis were all down-regulated.It is interesting that the flagellar assembly genes were up-regulated in this stage.This is consistent with previous report that the Pseudoalteromonas species could swim away to find new nutrients when the local food is depleted.For Y09,a striking feature was that 13 transporter genes for adsorbing glycine betaine,proline,oligosaccharides and amino acids were up-regulated in this stage.This indicated that when the nutrient concentration was decreased,Y09 could expressed more transporters to increase the substrates uptake efficiency.Finally,we proposed an interaction model between copiotrophs and oligotrophs in open surface seawater:when HMW substrates arrived,the copiotroph responds firstly and secrete extracellular enzymes to degrade HMW substrates;the copiotroph can adsorb the degraded nutrients efficiently and grow quickly while the oligotroph can only maintain survival;when the nutrients are depleted,the copiotroph decay quickly and oligotroph can utilize the cell lysate of copiotroph to grow.The growth of oligotroph was after the growth of copiotroph and accompanied with the decay of copiotroph.This can result in the oscillation coexistence of copiotroph and oligotroph.This dissertation showed the microbial community of the ocean and polar ecological environment using metagenomic technology.We compared biogeographic distribution patterns of bacterial community in South China Sea,reconstructed the functional ecosystem in the Antarctic Union Glacier region,and explored the heterogeneity in bacterial community compositions of southwest and central Indian Ocean.The importance of microorganisms to global chemical cycles was revealed.In addition,we studied the co-culture experiments of two bacteria with distinct nutrient types-Pseudoalteromonas sp.SW0106-04 and Brachybacterium sp.SW0106-09,proposed the co-existence model of copiotrophs and oligotrophs in surface seawater,and revealed the interaction mechanism.