Phosphorus Stress Promotes Mineral Weathering Of Pseudomonas Azotoformans F77 And The Mechanisms Involved

Mineral weathering is one of the most important environmental process that affects the compositions of soil.The mechanisms of interaction between microorganisms and minerals have been extensively studied.At present,bacteria with silicate mineral weathering ability are extremely rich,however,the mechanisms on silicate weathering have still not been understood,especially the mechanisms of bacterial phosphorus stress in the weathering process of silicate minerals has not been reported.Therefore,study on the mechanisms of weathering silicate minerals under phosphate stress will provide theoretical and experimental bases for the mechanisms involved in the interaction between silicate mineral and bacteria.The mineral-weathering bacterium Pseudomonas azotoformans F77 was used to study the physiological characteristics and the molecular mechanisms of mineral weathering.The concetrations of Fe and A1 in culture medium are used as the indicators of mineral weathering.The initial phosphorus concentration in the BHm is 980 μM.When biotite was used as the test mineral,the selected phosphorus concentration was 25 and 50 times lower than the initial phosphorus concentration in the BHm,respectively.Since feldspar is a phosphorus-free mineral,the the selected phosphorus concentration was 10 and 100 times lower than the initial phosphorus concentration in the BHm,respectively.Due to the poor growth of the strain at the low phosphorus conditions,comparison of the released elements and produced organic acids by the strains was made besed on the number of 105 cfu mL-1,the results show that the released elements and produced organic acids were significantly higher in low phosphorus than in normal phosphorus.The relative expression of the genes in the strain was analyzed by qRT-PCR,and the expression of phosphorus stress genes in the low phosphorus with mineral was found to be up-regulated.The complete genome sequencing analysis of strain F77 showed one independent chromosomes of 2.95 Mb and the genes(clusters)related to phosphours stress were found.Two key genes of phoR-phoB and pstS were found in the chromosome.Mutants F77△phoR-phoB and F77△pstS were constructed via homologous recombination.The concentrations of mobilized A1 and Fe were decreased by 32-52%during the biotite weathering process in the presence of the mutant strains F77△phoR-phoB compared to strain F77.Furthermore,gluconic acid production and the number of adsorbed cells on the mineral surface of the mutant strains decreased by 4.3-6.7%and 25-28%respectively compared to strain F77.The concentrations of mobilized Al were decreased by 2.4-45%during the feldspar weathering process in the presence of the mutant strains compared to strain F77.Furthermore,gluconic acid production and the number of adsorbed cells on the mineral surface of the mutant strains decreased by 2.3-3.4%and 8.9-15.7%,respectively,w-hen the genes phoR-phoB were complemented into the mutants,no significant differences in Al and Fe released from biotite were found.Comparative proteomic analysis of wild strain F77 and the mutant strain F77△phoR-phoB by iTRAQ technology,and the protein regulated by phoR-phoB were further found.The results showed that 20 proteins were up-regulated and 35 proteins were down-regulated.Based on protein function annotation,among these differential abundance protein,some of them were found to be related to stress response,energy synthesis,metabolism,transport,biosynthesis,cellular structure,transcription,and unknown functions.Protein-protein interaction was used to identify phoR-phoB-regulated proteins and significantly enriched KEGG pathways involved in regulated proteins,it was found to be involved in oxidative phosphorylation,pentose phosphate pathway,two-component system pathway,etc.The function of the regulated part of the protein was analyzed.The expression of the biguanylate cyclase wspR,which controls the function of bacteria,was down-regulated,and the ribosome phosphorase kinase prs,which inhibits oxidative stress,was up-regulated,and the ppk was down-regulated in the process of bacterial organic acid metabolism.The diguanylate cyclase wspR,which may be involved in the mineral weathering process,affects the production of bacterial extracellular polysaccharide,was down-regulated(1.38 times).The expression of ppk was down-regulated(2.34 times)in bacterial organic acid metabolism.The results showed that phoR-phoB could affect mineral weathering by regulating the expression of the genes responsible for organic acid and extracellular polysaccharides production,adhesion,and flagellar movement of the strain.