| In microorganisms, although iron is a necessary element for cells,it’s toxic to cells when microorganisms absorb inordinate number of iron. Thus, microorganisms developed many methods to order the iron homeostasis. In Gram-negative bacteria, the transport, store, and usage of iron are regulated by the ferric uptake regulator(Fur). Fur can interact with ferrous and form a dimer. The expression of fur is affected by the iron content and a sigma factor RpoS. It was reported that Fur can influence the colonization and infection of pathogenic and rhizospheric microorganism. Pseudomonas stutzeri A1501 is an associative nitrogen-fixing bacterium, whose effective nitrogen fixing ability and root colonization need copious iron-containing proteins, particularly the absorption and regulation of ferritin. There is a fur gene in the genome of A1501, which is highly homologous with Pseudomonas aeruginosa. In this study, we constructed the mutant of fur, analyzed the transcriptome and EMSA to explore the mechanism of Fur in transport of iron and nitrogen fixing. The main research results are described as follows:(1)In A1501, the expression of fur can be affected by iron content in the environment and RpoS. By qRT-PCR and Western blotting, under iron-starved condition the expression of fur increased doubled compared with normal condition, and in excessive iron condition the expression of fur was half of the normal condition. We found that in rpoS mutant strain, both of gene and protein expression of the fur were 2-fold than that in wild type. Moreover, we found the possible binding site of RpoS protein at the promoter of fur. However, the RpoS protein cannot be expressed successfully. The relationship between fur and rpoS needs more experiments to demonstrate.(2)Fur participated in the regulation of iron transport in A1501. In LB medium with 16 μM iron, the growth of A1501 and fur mutant showed no significant difference, but the intracellular iron of fur was 1.3 folds than that in A1501. The analysis of genome found there were 28 iron absorption related genes, including two TonB systems, sidephore synthesis genes and ABC transport. The result of qRT-PCR shows that all of the genes were 8-fold up-regulated in fur mutant. The hereinbefore indicates that Fur in A1501 played a negative-regulated role in the iron transport system. Furthermore, we found fur could inhibit the expression of a small RNA prrF, which can affect the stability of sodB mRNA. The result of the experiment was verified by EMSA.(3)Fur can influence the expression of nitrogen fixing-related genes in A1501.The nitrogenase activities of fur mutant was only 10 percent of A1501. By qRT-PCR and Western blotting, we found the expression of nifHDK was down-regulated. By analyzing the transcriptome, we found 48 genes in the nitrogen fixation island were down-regulated. However, the genes of general nitrogen metabolism regulation system, such as rpoN, ntrBC and glnD, had no significant difference. We analyzed the promoter of nifH and hesB and found the possible binding sites of Fur. EMSA showed Fur protein can bind to the promoter of nifH.All the above showed that Fur is a transcriptional regulation factors and can bind to DNA sequences. The expression of Fur is affected by iron and RpoS. Fur regulates some metabolic pathways in A1501, including negative regulation of iron transport and positive regulation of nitrogen fixing-related genes. |
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