| The process of using microorganisms to decompose metallic sulfide minerals is called as bioleaching,it's a friendly environmental bioleaching technique.Acidithiobacillus caldus(A.caldus)is a kind of obligated autotrophic gram negative bacteria,which grow at low pH(<3),and usually widely distribute in the Acidic hot springs and sulfide deposit.A.caldus is one of the dominant bacteria in the bioleaching.It has received the widespread attentions for its high-efficiency sulfur metabolism and significant application value in industry.There is many difficulties in the industry application of A.caldus,because of its long generation time and lower growth yield,especially the strong inhibitory effects for high iron and low pH conditions in the later bioleaching period.Therefore,it becomes more and more important to research the environmental adaption of A.caldus for its theoretical value and practical application.Ferric uptake regulator is the member of FUR superfamily,and has a wide range of homologue.Recent studies of different bacteria revealed that Fur,as a global regulator,plays important roles not only in iron uptake and storage,but also in a large number of "non-iron" functions,such as redox resistance,acid resistance,flagella synthesis and motility,cell respiration.The central role of Fur in the metabolism of prokaryotes through the regulation of plenty of genes involved in different functional categories in response to different environmental stimuli.In prokaryotes,feoB is the major function protein of the feoABC operon.The expression offeoB is regulated by the Fur,and the expression level offeoB is affected by the iron level of the habitat.However,the function of Fur and the regulation of FeoB in A.caldus are still unclear and the studies about the iron transport system in this bacterium are not be reported till now.Therefore,it is necessary to carry out the research on the iron transport system in A.caldus,which will bring new insights in the metabolic regulation network and mechanism of environmental adaptability about A.caldus.This article is the first report on the function offur and feoB in A.caldus,which contains the following parts:Firstly,we performed bioinformatic analysis of iron transport and regulation system in A.caldus.We aligned amino acid sequences of Fur and FeoB from A.caldus and other bacteria in Acidithiobacillus to illustrate their evolutionary relationship.The result shows that A.caldus had a closer relationship compared with other bacteria in Acidithiobacillus.In addition,we used the software online to simulate the protein structure of Fur,found that Fur had two metal binding site,which i5 similar with E.coli.Then we also predicted the transmembrane structure of FeoB,and found that FeoB has 10 transmembrane helixs,showing the similar features to FeoB in other bacteria.Secondly,we successfully deletedfur andfeoB genes in A by using markerless gene knockout technology and obtained the mutant strains Afur and AfeoB.Then we constructed a plasmid containing the fur gene under the transcription of tetH promoter,and conjugated the generated plasmids in the mutant and wild type of A.caldus to constructed the complement strain ?fur(pJRD215-PtetH-fur)and overexpression strain WT(pJRD215-PtetH-fur),respectively.Thirdly,we analyzed the growths of these constructed strains.When the strains cultivated in the different initial pH of culture medium,no obvious difference was detected between the mutants and wild type in the earlier growth stage,but the mutants(?fur and ?feoB)showed lower growth levels than that of wild type entering the late logarithmic and stationary phases;the complement strain?fur(pJRD215-PtetH-fur)showed the similar growth characteristics to the wild type;the growth difference between the overexpression strain WT(pJRD215-PtetH-fur)and the wild type appeared in the late logarithmic growth phase,such as the later time of entering the stationary phase and the higher growth level for the fur overexpression strain.Therefore,we supposed that Fur and FeoB probably play roles in the late growth stage with the low pH value.Finally,the transcriptional levels of related gene in the different strains cultivated in Starkey-S0 medium were tested by using Real-time qPCR.Results showed that Fur not only regulated iron uptake system gene(including ferrous and ferric uptake system)and iron storage protein,but also affected the transcription of the SOS-response repressor,and molecular chaperone dsbG.It was indicated that Fur plays important roles in the metabolic process modulation and environmental adaption.Moreover,the expression of iron uptake and storage protein genes in AfeoB showed obvious transcriptional changes.It was worth noting that the expression of manganese and iron superoxide dismutase also up-regulated,which indicated that iron has key role in intracellular redox regulation.This work analyze the biological function of Fur and FeoB,provide the basic knowledge to further identify the Fur-regulon,and reveal the regulatory mechanism and the regulatory network,and has the theoretical value and practical application. |
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