Screening And Identification Of Manganese Tolerance Genes Of Bacillus Safensis

Manganese is abundant in nature and is one of the essential trace elements for living organisms.However,the high concentration of manganese will cause the pollution of the ecological environment and the destruction of manganese balance in the body,which will lead to poisoning and affect the health of animals and human beings.Manganese oxide bacteria mainly exist in the fresh water,oceans and soil environment,the main role is to Mn(?)into Mn oxide(?)and Mn(?),the biological oxidation process of the non-biological oxidation process has the characteristics of fast,is a major cause of manganese oxides formed in the environment.The manganese oxidation mechanism of a few species of marine bacteria has been mainly studied at home and abroad,but the related research on soil manganese oxidizing bacteria is poorly understood.Bacillus safensis strain S7 is a bacterium with a manganese tolerance capacity of 2200mg/L isolated from the soil in the manganese smelting area.The solid and liquid oxidation was measured by LBB reagent.At the same time,the bacteria cultured without manganese and with manganese were made into electron scanning electron microscope.It was observed that the bacteria cultured with manganese had obvious oxide attachment on the surface,so it was inferred that the strain did indeed have manganese oxidation activity.On this basis,we chose to use transcriptomics(RNA-seq)technology to compare the differential gene expression of Bacillus safensis in the logarithmic growth phase and the stable growth phase under the treatment of manganese stress concentration of 0mg/L and 250 mg/L In this case,the culture time was 8 h,12 h,16 h,and 24 h for a total of 8 transcriptomes.It is hoped that the genes related to manganese tolerance will be screened out,which will lay a theoretical foundation for the study of bacterialmanganese oxidation mechanism.The results obtained are as follows:1.The manganese oxidative activity of Bacillus safensis is 7.75 ?mol/(L.d).2.The data obtained by sequencing were compared by STAR and counted by featurecounts.Finally,DESeq2 and edgeR were used to analyze the differential expression of genes.In the logarithmic growth period,a total of 1199 genes with significant differences,502 up-regulated genes and 538 down-regulated genes were obtained.During the stable growth period,1462 genes with significant differences,760 up-regulated genes and 702 down-regulated genes were obtained.There were 518 differentially expressed genes shared in the two periods.Compared with the control group,differential genes in the logarithmic growth phase are mainly enriched in flagella synthesis,TCA cycle,and oxidative phosphorylation pathways;differential genes in the stable growth phase are mainly enriched in metabolism,amino acid biosynthesis,and antibiotic biosynthesis,There are more enriched non-ribosomal peptide structures,valine leucine and isoleucine biosynthesis,niacin and nicotinamide metabolism,streptomycin biosynthesis and C5-branched chain binary Acid metabolism and other pathways.These pathways may be regulated at different growth stages of bacteria to deal with manganese stress.3.16 genes were randomly selected from the two groups of differential genes,including: 6 genes related to amino acid metabolism,5 genes related to oxidative phosphorylation,1 flagellar gene,1 gene related to glycolysis,and 1 two-component system Related genes,one manganese oxidase gene and one copper transporter gene.Fluorescence quantitative PCR was performed on 16 genes at different manganese stress concentrations and different incubation times.The experimental data showed that they were consistent with the transcriptome analysis results,and the analysis results were true and reliable.The expression of the 16 genes did not have the same pattern.Under different manganese stress conditions,the expression of the genes had a temporal change.Among the genes related to amino acid metabolism,except for the expression pattern of gene3997,which tends to be flat,the other genes have the largest expression at 4-8h,and gradually increase after 16h;the oxidative phosphorylation gene basically maintains a stable low expression in the first 4h,reaching at 8h Peak,there is a sharp decline in 8-16 h.The gene expression in the high concentration manganese treatment was mostly higher than that in the low concentration manganese treatment.Most genes peaked at 8h,showing a bimodal expression pattern.4.Gene601 was knocked out by homologous recombination single exchange method,and the manganese oxidation ability of the gene601-deficient strain was lower than that of the wild strain.It is proved that gene601 is a key gene for manganese oxidation of Bacillus safensis.