Effects And Mechanisms Of Soil Bacterial Interactions On Community Structure And Succession

Soil is one of the most complex ecosystems on the earth.Healthy soils facilitate the maintenance of ecosystem services vital to life.As the most abundant and active microbial taxa in the soil,soil bacteria play a key role in soil health,plant productivity and ecological security.Bacterial interactions,including cooperation and competition,determine not only the structure,diversity and stability of bacterial communities,but also their function,resulting in communities exhibiting emergent properties.Dissecting bacterial interactions can help us to better understand and utilise soil microbial communities.Due to the complexity of the soil environment and the diversity of soil bacteria,it is not clear about the influence of bacterial interspecific interactions on community structure and function under the combined effect of multiple factors and the molecular mechanisms of interspecific interactions driving community succession.In soil environments,interactions between bacteria occur more at the community level,and the phenomenon of community coalescence is less well understood and appreciated.Therefore,this paper explores the ecological roles and mechanisms of soil bacterial interspecies interactions in both simple synthetic communities and complex soil environments.Two model bacteria,Escherichia coli K12 and Pseudomonas putida KT2440,were used to construct synthetic communities with different initial ratios.The synergistic effects of initial inoculation ratios and carbon source types on the structure of synthetic communities and bacterial interspecific interactions were investigated by fluorescence quantitative PCR,Biolog ecological panels and other molecular biology techniques;the potential molecular mechanisms of community convergence driven by bacterial interactions were elucidated by transcriptome sequencing;the dynamics of soil bacterial community coalescence and the effect of community-level interactions on soil bacterial communities were investigated using amplicon sequencing based on soil microcosm experiments.The main results are as follows:（1）The synergistic effect of the initial ratio and carbon source types on community structure,function and bacterial interspecific interactions was clarified.Under most carbon source conditions（59/71）,the final composition of the synthetic community depended on the initial inoculation ratio of the strains,and this effect was limited by the strain’s preference for carbon source,suggesting that the initial inoculation ratio and nutritional conditions synergistically regulate the final structure of the synthetic community.The initial ratio of strains also affects the metabolic capacity of the synthetic community.Of the 14U2 carbon sources for which both E.coli K-12 and P.putida KT2440 were unsuitable,the synthetic community can only make effective use of these carbon sources when the initial ratios of the strains are 1∶1 and 1000∶1,while the strains do not make good use of these carbon sources when the initial ratio is 1∶1000.Using carbon utilisation efficiency as an assessment factor,a new model for defining interspecific interactions was proposed,and the interaction patterns of carbon source utilisation in co-cultures was evaluated.The results showed that synthetic communities with an initial ratio of 1∶1 had the most positive effects,while synthetic communities with an initial ratio of 1∶1000 had the strongest negative effects.Among the 14 carbon sources in the U2 group,almost all co-cultures with initial ratios of 1∶1 and 1000∶1 had positive interactions,which may be the main reason for the high metabolic capacity of bacteria under this ratio.The results provide a deep understanding of the microbial interaction mechanisms and provide a scientific basis for regulating the soil microbial community using a synthetic microbiome.（2）The potential molecular mechanisms of community convergence driven by bacterial interspecies interactions were revealed.After co-culture of E.coli K-12 and P.putida KT2440 at different ratios for 24 h,the community structure and species gene expression converged.There are three mechanisms for the regulation of gene expression by species during community convergence.First,the“zero-time effect”,whereby species interact upon contact,with gene expression being strongest at 8 h,and the effect on community structure being felt later in the culture.Secondly,“population effect”,whereby species that are in the majority in a synthetic community have a more pronounced effect on species that are in the minority.In the“1∶1000”co-culture system,the differentially expressed genes of the minority E.coli K-12 were significantly greater than those of P.putida KT2440,whereas in the“1000∶1”co-culture system,the differentially expressed genes of the minority P.putida KT2440 were significantly greater than those of E.coli K-12.Finally,the“tightly regulated effect”,the community convergence due to interspecific interactions was strictly regulated by time and the initial ratio of strains.In particular,40 of the 63 metabolic pathways were continuously inhibited throughout the convergence process,mostly related to carbon metabolism,fatty acid metabolism and secondary metabolite biosynthesis,16 were conditionally activated,and the only 7 pathways that were continuously activated were related to amino acid biosynthesis,RNA degradation and bacterial secretion systems.The above findings suggest that gene expression changes induced by bacterial interspecies interactions play an important role in community succession,and the gene regulation patterns provide a molecular basis for subsequent insights into the bacterial community convergence.（3）The dynamic process of soil bacterial community coalescence and the effect of community-level interactions on bacterial communities were elucidated.The results of microcosm experiments showed that inoculation of exogenous bacterial community into unsterilised soil had no significant effect on soil physicochemical properties,whereas inoculation of exogenous bacterial community into sterilised soil significantly increased soil p H and NH₄⁺-N and decreased NO₃^--N.The majority of species in the exogenous bacterial community died out after 7 d of inoculation into unsterilised soil,whereas more species colonized successfully after inoculation into sterilised soil.Community coalescence had no significant effect on theαdiversity of the soil bacterial community,but significantly altered the community structure.The significant change in community structure was not due to colonization by exogenous bacterial community,but probably to a“legacy effect”.The coalescence bacterial co-occurrence network was more complex,with more keystone species and increased negative connectivity between species,presumably due to competition between the exogenous and resident bacterial community resulting in more negative interactions.Soil NO₃^--N was significantly associated withα-diversity and composition of coalescence communities.ASVs were classified as“resident”,“invader”,“shared”and“unknown”according to whether they were present only in exogenous bacterial community or resident microbial communities.The 7 ASVs with significantly increased abundance in the coalescence community,all of which belonged to the“invader”and“shared”,were not strongly associated with environmental factors,while showing a higher correlation with the top 50 ASVs in“resident”.These high abundances of resident bacteria may be potential“helpers”of invasive bacteria.The above results provide a new way of studying the soil bacterial community coalescence,and provide theoretical guidance for further studies on soil community coalescence and soil microbial community transplantation.