Isolation,Identification,Genome Analysis And Application Of Heavy Metal Tolerant Strain Mucilaginibacter Rubeus P3 Sampled From Zijin Mining Area

In this study,we collected 43 soil samples from the Zijin gold-copper mine located in Caixi Town,Shangxian County,Shanghang County,Fujian Province.We isolated and further characterized strain Mucilaginibacter rubeus P3?M.rubeus P3?that displayed high resistance to arsenic?As?,cadmiun?Cd?,zinc?Zn?and copper?Cu?.We attempted to better understand the potential resistance mechanisms to these heavy metals?metalloids?via whole genome sequencing;We also demonstrated the plant growth promoting effects of M.rubeus P3 on stimulating the growth of rice plants?Ri ben qing?subjected to arsenite[As???]stress in a greenhouse.In addition,we wanted to explore the possibility and prospects of using this strain to remediate contaminated soils.Screening for heavy metal resistant strains.?1?Screening of strainTo screen for heavy metal-resistant strains,we applied a soil sample diluted with PBS to R2A solid plates containing 2 mM,4 mM,8 mM Cu²⁺,and isolated 19 Cu²⁺tolerant bacteria,named C8,C13,C14?M.rubeus P3?,C16,C18,C22,C23,C24,C25,C27,C29,C33,C34,C35,C39,C41,C42,C43 and C50,of which C13,C14,C41 and C42 colonies were pink.The morphology between these strains was similar and this could be further substantiated by single-cell SEM.The subsequent plant growth-promoting experiments found that the plant growth-promoting effect of C14 was the most significant.Therefore,C14 was further characterized in follow-up studies.It could be determined that the Minimum inhibitory concentration?MIC?of strain C14 on Zn²⁺,Cu²⁺,As³⁺and Cd²⁺were 21 mM,3.5 mM,4.5 mM,0.2 mM,respectively.?2?Identification of strainIt could be shown that C14 belonged to the Actinobacteria phylum Sphingobacteria Class,sphingobacteriales Order,Sphingobacteriaceae Family,Mucilaginibacter genus by 16S rRNA comparative analysis.Phylogenetic tree analysis based on 16S rRNA found that Mucilaginibacter rubeus P3?C14?was closely related to Mucilaginibacter rubeus EF23^T?KX792140?,MucilaginibactergossypiiGh-67^T?jgi.1071184?,Mucilaginibacter gossypiicola Gh-48^T?EU672805?.The similarities of their respective 16S rRNA sequences were 99.93%,98.99%,and 97.55%,respectively.Furthermore,taxonomic and genomic analysis showed that C14 is a gram-negative,strictly aerobic,non-spore-forming and non-motile bacterium.?3?Genome analysis of the strainThrough data analysis based on whole genome sequencing?draft genome sequence and annotation?,we found that the genome size of C14was 7125794 bp containing 6190 protein-encoded genes,45 tRNA-encoding genes,and 3 rRNA-encoding genes.We further analyzed C14 and found that:·The strain C14 contains six genes?mco?encoding multicopper oxidases,which can use copper ions as a coenzyme to oxidize various organic substrates or inorganic substrates including Cu?I?.The gene products of the cusCBA operon encoding a three-component efflux system use methionine residues to bind and export Cu?I?or Ag?I?accumulated in the periplasmic space.·In addition,three operons encoding genes involved in arsenic resistance could be identified,namely arsNC₁R₁,acr3 and arsMC₂R₂,respectively.The expression of acr3 gene has been shown to confer resistance to arsenite[As???].·There are 10 genes related to zinc(Zn²⁺)and cadmium(Cd²⁺)tolerance,and contain a czc operon ensures for resistance to Cd ²⁺,Zn²⁺and Co ²⁺ions through a tricomponent export pathway,In cultures grown with high concentrations of heavy metals,czc-mediated efflux of cations could be followed by a process of metal bioprecipitation.·Meanwhile,it was discovered that C14 contained 27 genes putatively related to plant growth and extracellular polysaccharide secretion,genes pel and one psl may participate in biofilm formation.Our experiments found that C14 forms a biofilm on R2A solid medium and secretes a pink pigment.Therefore,the above results indicate that under heavy metal stress,C14 may bind heavy metals in vitro by producing abundant extracellular polymers?mainly mucopolysaccharide and capsular polysaccharide?.?4?Growth characteristics of the strainColonies of C14 were round,opaque,with a convex,smooth surface,smooth edges and moist.Cells of C14 were without flagella and rod-shaped,with a size of 0.3-0.4 um long and 1.1-1.3 um wide.Optimal medium was detected by testing different media and R2A medium was found to be the optimum medium for C14.The optimum growth temperature,optimum growth NaCl and optimum growth pH for strain C14 were 28°C,1.0%?w/v?,and pH 5.?5?Characterization of C14 plant growth promotionCompared to the control group without As???,different concentrations of As???significantly increased the IAA yield of C14,and the IAA yield of C14 was the highest under 1.2 mg L^-1 As???treatment.Phosphorus solubilization experiments showed that the strain grew vigorously on Inorganic phosphorus medium and generated a transparent phosphorus-dissolving circle,while no transparent dissolved phosphorus ring was produced on the organic phosphorus medium medium,indicating that C14 could dissolve inorganic phosphorus but could not dissolve organic phosphorus.Rice hydroponic experiments found:Different concentrations of As???stress inhibited the growth of rice roots,causing browning or blackening of roots.Under As???stress,inoculation of C14 could significantly increase the root activity of rice?the roots are whiter and thicker?.Different concentrations of As???treatment significantly increased the chlorophyll and N content of rice,and inoculation of C14 further increased the content of chlorophyll and N.However,the inoculation of strain C14 did not significantly increase the biomass,root length,plant height and other biological indicators of rice under As???stress,Therefore,the growth-promoting effect of the strain and preferred growth-promoting conditions require further experimental investigation and verification.In summary,in this study,we isolated a strain?C14?and identified its species and genus via 16S rRNA comparative sequence analysis.In addition,we investigated the growth characteristics and optimal growth conditions of C14 based on the results of genomic and physiologic analysis,and we obtained putative genes involved in resistance mechanisms to heavy metals?metalloids?of this strain.Finally,we conducted a hydroponic experiment to investigate the growth promoting effects of C14on a rice plant subjected to As.