Screening Of Lead-resistant Microorganisms In Nansi Lake Sediment And Study On The Molecular Mechanism Of Resistance

Background:Soil heavy metals can pass along the food chain and accumulate in organisms,posing a potential threat to human health.It is a scientific issue that cannot be ignored to control the soil contaminated by heavy metals and control its transmission along the food chain.Aims:Screen native anti-heavy metal microorganisms,identify strains,and analyze the resistance mechanism of strains to heavy metals.Methods:In this paper,a strain 1D with good lead resistance was isolated and screened from the bottom mud of Nansi Lake.It was identified as Bacillus cereus by16S r DNA sequencing and phylogenetic analysis;The surface morphology changes of the strain under the culture condition of lead(600mg/L);through transcriptome sequencing analysis and GO and KEGG enrichment analysis,the molecular mechanism of lead resistance of strain 1D was explored.Results:1.Screening and identification of lead-resistant strainsA strain that can grow well in a medium with a Pb²⁺concentration of 600 mg/L was selected from the sludge at the bottom of Nansi Lake.It was identified as Bacillus cereus,named Uncultured Bacillus sp.clone 1D,and Genbank accession number was MK583952.2.Ultramicroscopic observationObservation of the strains grown in the lead-free medium and the lead-containing(600mg/L)medium under the electron microscope found that compared with the strains grown in the lead-free medium,the cell surface of the strain 1D under Pb²⁺stress formed many nanometers particles,and the surface is covered with mucus.3.Transcriptome sequencing and analysis(1)Through Illumina sequencing,the BC0 group averaged 7,352247 Raw reads per sample;the BC6 group averaged 7,166,681 Raw reads per sample.After filtering the original data and quality inspection,we obtained clean reads that can be used for subsequent data analysis.The average number of BC0 group was 7,228,546;the average number of BC6 group was 7051033.After gene annotation,there are a total of 4379 genes between the BC0 group and the BC6 group,of which 83 are unique to BC0 and 112 are unique to BC6.After analyzing the difference in gene transcription abundance between the BC0group and the BC6 group,the total number of differential genes between the two groups was 649,among which the number of genes up-regulated by BC6 relative to BC0 was 359 and the number of genes down-regulated was 290.(2)After GO function enrichment analysis,compared with the BC0 group,the BC6 group has 76 functional groups that are significantly up-regulated,of which 62are in the biological process part,mainly oxidation-reduction process,drug metabolic process,interspecies interaction between organisms,ATP metabolic process and a series of genes related to phosphate metabolism process;there are 14 kinds of molecular functions,mainly oxidoreductase activity,electron transport chain related genes,ion binding related genes,ion transmembrane transporter activity and other functions.There are 4 functional groups that significantly down-regulate expression,namely,response to external stimulus,regulation of cellular process,biological regulation,regulation of biological process,all of which belong to the biological process part.(3)After KEGG pathway enrichment analysis,the genes up-regulated under Pb²⁺stress are related to 12 pathways,mainly oxidative phosphorylation,glyoxylate and dicarboxylate metabolism,citrate cycle,microbial metabolism in diverse environments,carbon metabolism and a series of amino acid degradation and metabolism pathways.The significantly down-regulated genes are related to three pathways,namely,bacterial chemotaxis,two-component system,and phosphotransferase system.Conclusions:Pb²⁺stress caused changes in the cell surface of strain 1D,induced strong oxidation-reduction reactions and respiratory metabolism in the cells of strain1D,stimulated active energy metabolism in the cells,and may force strain 1D to directly absorb a large number of amino acids from the environment for utilization.The genes related to binding and transport function played an important role in the 1D lead tolerance of the strain.At the same time,the expression of a large number of membrane-related genes in strain 1D was changed under Pb²⁺stress.We speculated that Pb²⁺might induce some changes in membrane-related functions of strain 1D,and achieve the purpose of keeping lead ions out of the cell through more precise control of material entry and exit.In addition,we also observed down-regulated expression of genes encoding mar R transcription factors,suggesting that in the anti-lead mechanism of strain 1D,it seems to be more likely to eliminate the harm caused by lead through some metabolic processes in the cell than to extract lead ions.