| Bacteriophages(phages)can be divided into lysogenic phages Chronic phages,and virulent phages according to their lifecycle.The fusion of genetic material provides an opportunity for the evolution of mutualism between the lysogenic phage genomes and their host.Studies based on marine ecosystems have shown that lysogenic phages among phages are inclined to develop mutualism with their hosts to resist unfavorable environments,this phagehost interaction named protective parasitic behavior of lysogenic phages.However,our understanding of the protective parasitism of lysogenic phages in soil environments relevant to human beings is far from sufficient.A full understanding of how protective parasitism of lysogenic phages acts in different environments can expand our basic knowledge of phage populations and deepen our understanding of bacteria-phage interactions in nature environments.Arsenic(a class of toxic metalloids)that are widely distributed in soil and threaten the survival of soil microorganisms.In order to resist arsenic toxicity,microorganisms have evolved a complex anti-arsenic system,and long-term research has made us have a more systematic understanding of the microbial anti-arsenic system,so arsenic-contaminated soil is an ideal window to investigate the protective parasitism of lysogenic phages.Therefore,this paper investigated the protective parasitic behavior of lysogenic phages in arseniccontaminated soils sampled from an arsenic-contaminated area to address the gap in the current understanding of bacterium-phage interactions in the soil environment.The research work and results of this paper include the following four sections:The 1st section studied the distribution of lysogenic phages in arsenic-resistant bacteria(ARBs)in different arsenic-contaminated soils and the abundance of arsenic-resistant genes(ARGs)in lysogenic phages.Using specific primers,the occurrence of ARGs in ARBs and lysogenic phages genomes was verified by real-time quantitative polymerase chain reaction(RT-q PCR);The correlation between the abundance of ARGs in the lysogenic phage genome,the arsenic background concentration,and the abundance of ARGs in ARBs was further investigated;Finally,the diversity of ARGs carried in lysogenic phages was also analyzed.The results showed that the soil arsenic background concentration was negatively correlated with the microbial biomass but the number of lysogenic phages was less affected by the arsenic concentration.The abundances of arsC and arsM in bacterial genomes and phage genomes increased with the increase of arsenic concentration,and were positively correlated with the concentrations of total arsenic,phosphate-extracted As(III)and phosphate-extracted As(III)in soil.Normalized abundance of ARGs in phages(normalized to gene copies per phage particle)and normalized abundance of ARGs in ARBs(normalized to copies pern 16 S r RNA).The correlation analysis showed that the horizontal gene transfer(HGT)of ARGs involved in phage was closely related to the establishment of bacterial arsenic resistance system.In addition,it was found that the arsM in phage genome has a higher diversity than the arsC.The 2nd section investigated the effects of flooding-driven rise in arsenic toxicity on active bacterial and phage communities in soil.A flooded microcosm reactor was inoculated with arsenic contaminated soil,and the soil geochemical properties,active microbial community(based on 16 S r RNA sequencing),phage populations were monitored during a 15-day flooding period.The results showed that the arsenic chemical speciation changed greatly due to flooding,mainly due to the large increase in the concentrations of dissolved As(III)and phosphoric acidextracted As(III),and the main driving factor for this increase was the redox potential(decrease of Eh)has little correlation with p H,total organic matter,total nitrogen,total phosphorus and other factors.The increase in As(III)toxicity significantly altered the composition of active microbial communities,both at the phylum and genus level.The number of bacterial species and the alpha diversity of the bacterial community varied with the duration of flooding,and they decreased in the first 5 days and recovered in the subsequent reactors,indicating that the microorganisms are highly adaptable to the toxicity of arsenic.Among the environmental factors,Eh has the greatest impact on the active microbial community,while As(III)among different arsenic chemical speciation has the greatest impact on the active microbial community,and both dissolved As(III)and phosphate-extracted As(III)have significant effects on the active microbial community.Phage populations also changed dramatically in the flooding period.In the first 2 days we observed a large burst of phage numbers,in which lysogenic phages were induced by As(III)to produce a large number of new virions,we also found that the number of lysogenic phages in the 15 th day of the microcosm was higher than that in the 0-day group,suggesting that activation and re-infection of lysogenic phage are associated with bacterial community evolution.The 3rd section investigated the substantial contribution of lysogenic phage-mediated transfer of ARGs to host adaptability of arsenic toxicity.The differences of gene expression in microbial communities with respect to functions(e.g.,HGT)were first analyzed by function prediction;Then,quantitative analysis of the arsM(selected because it is closely related to As(III)metabolism)in phage population were performed;Finally,the contribution of phagemediated lateral transduction of arsM to the increase of arsM abundance in microbial genomes before and after flooding was analyzed,and the resulting arsenic methylation ability in soil microbial communities was investigated.The results indicated that the bacterial community had active expression of functions related to signal transduction and cell synthesis during flooding period.The large differences in the abundance of genes expressed by the active microbial community for the genetic elements associated with HGT indicate that HGT between different bacteria is greatly enhanced with the increase of arsenic toxicity.The bacteria expressing oxidative stress tolerance genes had higher expression levels in first 2 days before flooding but decreased significantly after the 5th day,indicating that the bacteria quickly adapted to this extreme environment.Phage-mediated horizontal transduction of arsM contributed greatly to the acquisition of arsM by bacteria,and soil bacteria acquired elevated arsenic methylation ability after flooding.The 4th section introduced the ex-situ infection ability of ARG-bearing lysogenic phages and relevant influencing factors.The changes in the abundance of ARGs in the soil and the changes in the arsenic chemical speciation in the reactor were investigated by inoculating lysogenic phages to the soil of Mount Yuelu.The results showed that the abundance of ARGs in the soil microorganisms from Mount Yuelu inoculated with lysogenic phages from SM was increased,but different ARGs showed different growth patterns.After inoculation and culture,the abundance of arsC in soil microorganisms was negatively correlated with the background As(V)concentration and positively correlated with the background As(III)concentration;the arsM abundance in soil microorganisms was positively correlated with the background As(III)concentration.Likewise,the inoculation of lysogenic phages carrying ARGs changed the arsenic chemical speciation in the reactor.The increase in the abundance of the arsC increased the As(III)content,and the increase in the abundance of the arsM caused the production of arsenic methyl compounds in the reactor.This thesis systematically investigated the protective parasitic behavior of lysogenic phages in arsenic-contaminated soil,which provided considerable valuable information for understanding phage populations in soil environments,and for understanding bacterium-phage interactions in contaminated soils,which also contributed to our comprehensive understanding of phage populations in the environment. |
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