The Molecule Mechanisms Of Biotite-weathering Mediated By A Highly Effective Mineral Weathering Bacterium Pseudomonas Azotoformans F77

Biological weathering of silicate minerals is a widespread geochemical process,which has important impacts on soil formation and fertility,element geochemical cycles,and long-term atmospheric environment.At present,there are more and more studies on bacterial silicate mineral weathering,but the details and the molecular mechanisms involved in bacterial mineral weathering are poorly understood.This study investigates the molecular mechanisms of bacterial silicate mineral weathering,and explores the importance of acidolysis,adsorption,and acid tolerance of bacteria in silicate mineral weathering,which can provide theoretical and experimental basis for elucidating the molecular mechanisms of the interaction between bacteria and minerals.Biotite is used as the test mineral in this study.One highly effective mineral weathering bacterium F77 was selected from 15 strains which can produce gluconic acid.Further research was performed as following aspects:1)Compared with strain P1,Pseudomonas sp.F77 was more effective mineral weathering bacterium.By comparing the growth and metabolism of strain F77 with or without biotite,we found that biotite could promote the growth of strain F77 and the production of gluconic acid.2)The complete genome sequencing analysis showed that strain F77 was Pseudomonas azotoformans.RNA-seq was used to compare the gene expression level of the strain F77 with that of the strain P1 during the biotite weathering.The result showed that 2500 gene families are unique to strain F77,and 1895 gene families unique to strain P1.Annotating unique genes to the KEGG pathway,we found that the numbers of unique genes involved in membrane transport,cell motility and chemotaxis,carbon metabolism,and energy metabolism in strain F77 were significantly higher than that in strain P1.In carbon metabolism,the numbers of unique genes involved in citric acid cycle,amino sugar and nucleotide sugar metabolism,pentose phosphate pathway,fructose and mannose metabolism pathways were increased significantly in strain F77.Moreover,RNA-seq was used to compare the gene expression levels of strain F77 in the absence and presence of biotite.The results showed that 1284 genes were up-regulated,and 452 genes were down-regulated in the presence of biotite.Based on genes annotation,GO and KEGG pathway enrichment analysis,these differently expressed genes were found to be related to signal induction,substance transmembrane transport,energy metabolism,and acid stress response mechanisms.Simultaneously,the genes related to gluconic acid metabolism were also expressed differently(gcd up-regulated 2.3-fold,gad up-regulated 11.3-fold,glk up-regulated 1.6-fold,gntP up-regulated 2.8-fold,pqqE up-regulated 1.9-fold,and pqqF up-regulated 1.6-fold).3)RT-qPCR was used to study the differential expression of the genes related to gluconic acid metabolism.It was found that biotite could promote the up-regulation of genes gcd(glucose dehydrogenase,up-regulated 8.0-fold at 24 hours),gad(gluconate 2-dehydrogenase,up-regulated 8.4-fold at 24 hours),glk(glucokinase,up-regulated 3.0-fold at 24 hours)and gntK(gluconokinase,up-regulated 6.9-fold at 24 hours).Single-gene mutant F77?gcd,F77?glk,F77?gntK,and F77?gad were constructed by using homologous recombination.Meanwhile,mineral weathering experiments were performed to explore the differences in weathering effectiveness.The results showed that only the mutant F77?gcd had significantly lower dissolution of Fe and A1 than the wild strain F77.A double-genes mutant strain F77?gcdAgad was constructed.The weathering ability,weathering mode and molecular mechanism of strains F77,F77?gcd,F77?god and F77?gcdAgad were compared.The adsorption on the mineral surface by the mutant F77?adh,the production of gluconic acid and the activity of glucose dehydrogenase of the mutant strain F77?gcd reduced compared with wild-type strain F77.The ability of each strain to weather biotite was F77?gcdAadh<F77?gcd<F77Aadh<F77.The relative expression of the genes gcd,gad,glk,gntk,adh,ecpD and fimA in strain F77,F77?gcd and F77?adh was analyzed by RT-qPCR.The results showed that the deletion of gcd would affect the metabolism of gluconic acid and stimulate the expression of genes related to adsorption,the deletion of adh would promote the expression of the genes related to gluconic acid metabolism.The gluconic acid production and strong adsorption were the main mechanisms involved in biotite weathering by strain F77.4)iTRAQ and RT-qPCR were used to analyze the proteome of strain P.azotoformans F77 under different pH conditions.In this study,141 significant differential proteins were identified and categorized into energy metabolism,cell response to stimuli,cell membranes,DNA repair,ATPase.All of significant differential proteins were up-regulated.Gene knockout and physiological and biochemical tests showed that resR(response regulator)and nueR(putative protein)can enhance biotite weathering by increasing the acid tolerance of strain F77.Compared with wild-type strain F77,H+-ATPase activity of mutant F77?nueR and F77?resR decreased by 24%-32%and 35%-80%,respectively,and membrane fluidity increased by 15%-27%and 33%-62%,the concentration of putrescine and the ratio of NAD+and NADH decreased by 37%-70%and 70%-91%in the mutant F77?resR,respectively.The mutant F77?nueR could affect the acid tolerance by affecting intracellular H+-ATPase activity and cell membrane fluidity.Mutant F77?resR could affect acid tolerance,by affecting the production of intracellular putrescine,H+-ATPase activity,NAD+/NADH,and cell membrane fluidity.It was also verified that resR had the ability to regulate acid resistance and mineral weathering of strain F77 by using heterologous expression.