Study Of Ferric Uptake Regulator Of Deep Sea Bacterium Shewanella Piezotolerans WP3

Deep-sea is a special ecosystem for its permanent coldness,high pressure, darkness,low in the nutrient and oxygen.The special metabolic characteristics of deep-sea microbes are determined by this special deep-sea ecosystem.It is important to study the physiological characteristics of deep-sea microbes for understanding special aspects of deep-sea microbes and exploitation of extreme microbial resource.The deep-sea bacterium used in this study was isolated from deep-sea sediments of 1914m depth in west Pacific.The phylogenetic analysis and molecular study shows that the bacterium is a psychrophilic and piezotolerant microbe with the term as Shewanella piezotolerans WP3.WP3 belongs toγ-proteobacterium and contains 55 cytochrome C genes,which are 13 more than Shewanella oneidensis MR-1.The strain's color and RT-PCR results of cytochrome C genes indicate that these cytochrome C proteins are expressed and function as provider of energy for WP3 even under aerobic condition.These cytochrome C genes are supposed to be important for its adaptation to sub-anaerobic deep-sea environment.The physiological iron homeostasis is important for all microbes for two reasons. First,iron is important.It is not only component of some proteins,such as isocitrate dehydrogenase,but also takes part in DNA metabolism,TCA recycle and glycolysis et al as the co-factor.And iron is also toxic.Fe²⁺ can produce highly reactive oxygen species through catalyzing Fenton Reaction.So keeping iron homeostasis is important for microbes.Second,WP3 needs more iron than other normal strains for it contains much more Cytochrome Cs which need more iron for their maturation,And energy producing while the deep-sea environment contains lower iron levels.The pH of sea water is higher than freshwater,a little higher than 7.0,and difficult for the dilution of Fe3+.So the bioavilable iron is less in deep sea than other environments.Different from iron,the concentration of SO₄^2- is high in deep sea environment.So,the deep sea environment contains less soluble iron and more SO₄^2-.It would be interesting to know how WP3 control iron homeostasis for such balance.It is reported that Fur act through diverse regulation ways as an intracellular iron homeostasis regulator.This regulator exists in most of microbes,so as in WP3.A fur coding gene-swp2938 was found in WP3's genome and knocked out to construct a mutant-Fur mutant.In our study,microarray technology was applied to study the functions of Fur of WP3.And functions of Fur were found do keeping the iron homeostasis,through comparing Fur/WP3 and WP3 with iron depletion/WP3 with iron repletion these two microarray results.Higher intracellular concentration of Fe²⁺ in Fur mutant was indicated by microarray results comparison.And this suspension was directly demonstated by intracellular Fe²⁺ concentration levels comparison between Fur mutant and WP3.The anaerobic respiration abilities were weakened in Fur mutant,and this is the first report which describes fur gene deletion impacts anaerobic respiration abilities of shewanella species.In our study,the genes' down regulation of both heme biosythesis pathway and cytochrome C maturation pathway were found.And both of them were possible to contribute to impaction of anaerobic respiration.Through a series of experiments and analysis,that the down regulation of cytochrome C maturation genes was the main factor leads to impaction of Fur mutant's anaerobic respiration abilities was demonstrated by our study.In our study,the contribution of sulfate assimilation to iron transportation in deep sea bacterium-WP3 was firstly studied.Genes in assimilation of sulfate pathway were all up regulated in Fur mutant,which catch up our interest.In order to test their relationship with WP3's intracellular iron homeostasis,genes in this pathway were studied.Two mutants were got through knock out cysG gene and cysN-D genes,and the physiological test indicated those genes were important for transportation of iron in WP3 strains.Cystine is important for transportation of iron,because it is the main substance for siderophores biosythesis.But bacteria used to get cystine through transformation of other amino acid or directly absorb it from culture,not through assimilation of sulfate.The importance of sulfate assimilation pathway in WP3 relates to its special living environment-deep sea ecosystem was suggested in our study.The low in nutrients and high concentration of sulfate in deep sea environment,determines WP3 can not get enough cystine through usual pathways,but sulfate assimilation.So producing cystine through the assimilation of sulfate is a special character of deep-sea adaptation for WP3.