Interaction Mechanism Of Phage And Bacterial Communities In Organochlorine Pesticide Contaminated Soils

The overuse of pesticides is the main reason for soil contamination,which has an undoubted effect on soil microbes.Recently,much attention has been paid to soil bacteriophage or phage communities.Phage infection could interfere with the bacterial metabolites such as energy equitation,stress resistance and compound transformation,thus effecting the biochemical cycle.However,less study has been focusing on the change of soil microbe under pesticide contamination,much less the phage-bacteria interaction.In this study,the phage-bacterial communities’ relationship under pesticide stress(clean,light contamination and heavy contamination)was explained from the aspects of the species and function diversity of bacterial and phage communities,as well as laboratory examination of phage auxiliary metabolic genes,based on a typical organochlorine pesticide contamination site located at Yangtze river delta area,China.The results are as follows:(1)Clean soils were dominated by Proteobacteria(34.7%),Acidobacteria(22.5%),Verrucomicrobia(13.4%)and Actinobacteria(12.2%).Conversely,the relative abundance of Actinobacteria(49.2%)and Proteobacteria(36.5%).On the genus taxa level,Bradyrhizobium(4.4%)and Rhodoplanes(1.1%)were abundant in clean soils while Bradyrhizobium(2.9%)、 Streptomyces(4.4%)、 Conexibacter(2.8%)、Nocardioides(2.4%)were abundant in contaminated soils.Pesticide degradation related bacteria,such as Beta-proteobacteria,Burkholderia,Streptomyces and Nocardiodes were enriched in contaminated soils(P<0.05).Alpha diversity analysis showed that bacterial community had lower richness but higher evenness in contaminated soils comparing with the clean soils(P<0.05).The community composition of clean and contaminated soils clearly differed from each other(P<0.05),while no clear difference between light and heavy contaminated soils.(2)Overall,19,292 phage contigs were recovered from all 9 soil samples,with5,387 and 13,905 contigs in clean and contaminated soils,respectively,which could be clustered into 1,916 distinct viral clusters(VCs).The phage communities had unique diversity and function characteristics which was not covered by public datasets.Across the 11% phage contigs that can be taxonomically classified,Caudovirales was the dominant order in clean and contaminated soils,which mainly consisted of the families Siphoviridae,Podoviridae,and Myoviridae.In addition,higher phage diversity(higher richness and evenness indices)was found in contaminated soils rather than clean soils(P<0.05),while no difference was observed between light and heavy contamination(P>0.05).(3)10,932 phage-host linkage,which link 19 bacterial phyla to 4,041 phage contigs based on t RNA,CRISPR spacer and JGI matching method indicated that phages were prone to infect the dominant bacteria in soils,such as Actinobacteria and Proteobacteria,and the predicted host records was positive related with the abundance in metagenomes.Among the 26 contaminated soil phages,54 phage-host linkage were found and 14 of which had extensive host matching with 23 bacterial groups,which indicated that broad-host phages were widely present in contaminated soil environment.(4)We also detected the association in the genetic functions harbored between bacterial and phage encoded genes.The abundance distribution of bacterial encoded metabolic genes was similar regardless whether they originated from clean or contaminated soils(P>0.05).It is worth emphasizing that kinds of functional genes,including carbon,nitrogen(e.g.,nor D),phosphorus(e.g.,pho D),sulfur(e.g.,cys K and cys H)and pesticide degradation genes(e.g.,pca GH,ALDH,EC.3.8.1.2)were harbored in contaminated soil phages,indicating that phage genes can cover a range of bacterial metabolic activities and pesticide degradation pathways.More abundant carbon genes(27 gene categories with relative abundance of 19.30%)was found in contaminated soils instead of in clean soils(14 gene categories with relative abundance of 2.53% in clean soils),and network analysis also indicated the strong association between carbon cycle and pesticide degradation.Furthermore,with the diversity and abundance of core functional genes decreased under pesticide stress(P<0.05),the diversity of phage auxiliary metabolic genes was higher in contaminated soils and the abundance increased significantly from light to heavy contamination compared to clean soils(P<0.05).(5)Biogenetic analysis showed that the phage encoded pca GH was closely related to Pseudomonas.The recombinant p ET-32a(+)-pca GH plasmid was introduced into E.coli Arctic Express for induction expression to test the protein activity of the protocatechuate 3,4-dioxygenase.The results showed that the concentration of protocatechuic acid(PCA)in the activated enzyme treatment was significantly decreased by 51.9% and 59.2% compared with the control at the reaction time of 3h and 6h respectively.HPLC analysis also showed that a smaller PCA peak at 7.5 min and additional peaks appeared between 2.5 and 5.0 mins in the activated treatment.Those results indicated the active function of phage encoded pesticide degradation genes.In conclusion,for the microbial community of organochlorine pesticides contaminated soil,this study not only explored the diversity and function of bacterial and phage communities separately,but also explained the bacteria-phage interaction.The results indicated that it is urgent to further explore the diversity of soil phages and the important role of phage auxiliary metabolic genes in assisting bacteria survive under stress,which provides data support and theoretical basis for exploring soil viral ecology and interaction mechanism with another organisms,and a promising way of phage therapy application to remediate organochlorine pesticide contaminated soil.