The Mechanisms Of Biotite-Weathering Mediated By Iron Response Regulators In A Highly Effective Mineral Weathering Bacterium Rhizobium Pusense S41

Mineral weathering is one of the most important geological processes on the earth's surface,which is the basis for the formation of various landforms and soil.Silicate weathering plays important roles in terrestrial ecosystems.There are many reports on the mechanisms of bacterial weathering minerals,however,the mechanisms on silicate weathering have still not been understood,especially the mechanisms of iron response regulator genes in the weathering of silicate minerals has not been reported.The highly effective mineral-weathering bacteria Rhizobium pusense S41 which can produce many exopolysaccharides and siderophores was selected to study the ability of biotite-weathering.The siderophores produced by strain S41 was isolated,purified and type tested.The results showed that,when glucose or sucrose was used as only C source in YN media,the elements(Fe,Al,Si)released and the production of expolysaccharides and siderophores were significantly higher than those supplemented with other carbon sources(13.7-65.1%).In YN media supplemented with(NH4)2SO4,the production of exopolysaccharides was slightly decreased while the production of siderophores was significantly increased and the element concentrations were higher than those with other N source.When the medium was supplemented with Fe3+,the exopolysaccharides produced by strain S41 was slightly lower,while the production of siderophores was significantly decreased(41.2-79.6%),so the dissolution of the mineral elements was significantly reduced.When the medium was supplemented with dipyridy,the expolysaccharides production was significantly increased in the early stage(1 d and 3 d)and the concentration of siderophores was significantly increased(25.8-39.5%),so the mineral weathering was promoted.The expression of the iron response regulatory genes fur,rirA and irr were assessed by qRT-PCR at two different time points(8 h,24 h)in YN medium supplemented with or without biotite.The results showed that fur,rirA and irr may be involved in mineral weathering process.Mutants S41?fur,S41?rirA and S41?irr were constructed via homologous recombination.The ability of biotite weathering by the mutants and wild-type strain were compared.The results showed that there were no significant difference in Fe,Al,and Si concentrations,the production of expolysaccharides,siderophores and the number of adsorbed cells on the mineral surface between the fur mutant and wild-type S41.As the siderophores production of the irr mutant was significantly lower than that of the wild-type S41,the dissolution of Si,Al and Fe was also significantly lower(Fe:11.2-46.4%,Al:6.9-16.8%,Si:4.7-13.2%).Although the number of adsorbed cells on the mineral surface of the rirA mutant was slightly lower than that of the wild type,the production of expolysaccharides and siderophores was significantly increased,and the mineral weathering ability was significantly enhanced(Fe:24.1-53.2%,Al:14.7-52.2%,Si:18.7-48.9%).When the genes rirA and irr were complemented into the mutants,no significant differences in the production of polysaccharide,siderophores and the contents of Si,Al and Fe released from biotite were found.Under iron-limited conditions,the production of siderophores and expolysaccharides of strain S41 increased significantly while the biofilm formation decreased slightly.The siderophores production of S41?irr was significantly lower than that of wild type strain under iron-limited conditions,while the siderophores production of S41?rirA was significantly increased under iron-sufficient condition.The intracellular iron concentration of mutant S41?rirA was higher under iron-limited and iron-sufficient conditions while the intracellular iron concentration of the mutant S41 ?irr was significantly lower than that of the wild type S41.Under iron-limited conditions,the motility of S41?rirA decreased significantly and under iron-sufficient conditions,the biofilm formation of S41?rirA decreased significantly.The qRT-PCR results corresponded to the above phenotypes.Under iron-limited conditions,some of the NRPS system genes involved in the synthesis of catechol-type siderophores,the genes involved in iron uptake and transferrin-iron transporter,the genes involved in iron-sulfur cluster synthesis and the genes involved in expolysaccharides synthesis were significantly up-regulated while the genes involved in iron storage were significantly down-regulated.Under iron-sufficient and iron-limited conditions,the gene rirA directly or indirectly negatively regulates the NRPS system while the gene irr regulates the expression of these genes positively.The gene rirA mainly regulates the genes involved in iron uptake and transferrin-iron transporter,the genes involved in expolysaccharide synthesis,and the genes involved in iron utilization negatively under iron-sufficient conditions,while irr positively regulates the expression of these genes mainly under iron-limited conditions.In contrast,rirA up-regulates the expression of iron storage gene under iron-sufficient conditions while irr negatively regulates iron storage gene under iron-limited conditions.