Mechanism Of The Regulation Of Nitrogen Fixation Mediated By The Ferric Uptake Regulator Fur In Pseudomonas Stutzeri A1501

Iron,an essential element for microorganisms,functions as a vital cofactor in a wide variety of key metabolic processes.Ferric uptake regulator?Fur?is the key regulator of iron metabolism in Gram-negative bacteria,playing an important role in the regulation of ferritin activity and microbe-host interaction.Pseudomonas stutzeri A1501 is a species of associative symbiotic nitrogen-fixing bacteria from rhizosphere,highly demanding iron as the participation of a large number of ferritin?such as nitrogenase?for effective nitrogen fixation and root surface colonization.Previous research in our lab found that the mutation of fur significantly down regulated the nitrogenase activity of A1501,suggesting Fur might be involved in the regulation of nitrogen-fixing gene expression,but the specific mechanism is not clear.In addition,non-coding RNA PrrF has been reported to regulate the expression of a ferritin SodB at the post-transcriptional level through RNA–RNA interaction.Whether ncRNA PrrF is involved in the regulation of nitrogenase expression in A1501 is of great significance and is worth of further study.In this work,the effect of Fur responding to extracellular iron was clarified by measuring the growth and nitrogenase activity of wild type A1501 and its derivative strains under various iron conditions;the mechanism of the Fur-mediated regulation of nitrogen fixation in A1501 was explored by transcriptome analysis,protein–DNA interaction and RNA–RNA interaction.The main results are as follows:The growth and nitrogenase activity of wild type A1501,fur mutant?fur and complementary strain com-fur were determined under iron-rich(basic K Meidum,50?M Fe³⁺or Fe²⁺)and iron-limited condition(K medium containing 0.5?M Fe³⁺or Fe²⁺).The results showed that there was no significant difference in growth,indicating that 0.5?M iron(Fe³⁺or Fe²⁺)could meet the needs of growth.Results in nitrogenase activity showed that the activity in A1501 and?fur in the medium with Fe²⁺was 30%higher than that in the medium with Fe³⁺,indicating that Fe²⁺might be more ideal for biological nitrogen fixation of bacteria.The nitrogenase activity of A1501 and?fur under iron-rich condition was about twice as high as that under iron-limited condition.The expression of Fur protein was detected by Western Blot,showing that the expression of Fur was up-regulated under iron-limited condition;qRT-PCR results also showed that fur gene was responsive to extracellular low iron level.The transcriptomes of wild type A1501 and?fur grown to logarithmic phase under ferric-rich(basic K medium,50?M Fe³⁺)and ferric-starvation?K medium contained excessive iron chelator?conditions were analyzed.The results showed that there were 146 differentially expressed genes?88up-regulated and 58 down-regulated?out of 4273 genes in?fur compared with wild type A1501 during growth in ferric-rich medium including the significantly up-regulated genes encoding TonB-dependent ferric uptake system TonB-ExbBD,cytochrome c maturation system?Ccm?and denitrification key enzyme Nir/Dnr/Nor.Under ferric-starvation condition,compared with A1501,there were 150differentially expressed genes?49 up-regulated and 101 down-regulated?out of all genes in?fur.For example,the expression of?^H-coding gene rpoH was significantly down-regulated.Analysis and characterization of promoter sequences indicated that there were conservative Fur binding sites?Fur Box?in the promoter regions of PST0819-exbB2-exbD2-PST0822,dnrS,nirH,ccmA,nfiS and rpoH,suggesting that Fur might directly regulate the expression of these genes.The molecular mechanism of the regulation of nitrogenase activity mediated by Fur was explored,including both transcriptional and post-transcriptional regulation.At the transcriptional level,Fur interacted with the promoter of nitrogenase ferritin gene nifH,positively regulating the expression of nifH.The interaction between Fur and nifH promoter was confirmed in vitro by EMSA.At the post-transcriptional level,Fur negatively regulated the expression of ncRNA PrrF.PrrF directly interacts with nifH mRNA and molybdenum-iron cofactor synthesis gene nifQ mRNA,affecting the synthesis and assembly of nitrogenase.There was a conserved Fur binding site within the promoter region of prrF.The interaction between Fur and prrF promoter region in vitro was confirmed by EMSA.Result of Northern Bolt suggested that the expression of ncRNA PrrF was negatively regulated by fur and iron.Putative binding sites were found both between PrrF-nifH mRNA and PrrF-nifQ mRNA by RNA interaction and secondary structure prediction.The interaction between ncRNA PrrF and two mRNA fragments in vitro was confirmed by Microscale Thermophoresis?MST?.To sum up,Fur participates in bacterial iron response by regulating its own expression at protein level,and might be directly involved in transcriptional regulation of genes related to iron transport,nitrogen cycle and heat shock resistance.In this work,the molecular mechanism of iron transporter directly involved in the regulation of biological nitrogen fixation at both transcriptional and post-transcriptional level was reported for the first time,laying a theoretical foundation for further analysis in regulatory network and environmental adaptation of nitrogen-fixing bacteria.