Physiological Characteristic Under Micro-aerobic Condition And Regulatory Analysis Of Dissimilatory Denitrification In Magnetospirillum Gryphiswaldense MSR-1

Magnetotactic bacterial?MTB?absorb large amount of iron to synthesize intercellular nano-scaled magnetosome only under low oxygen condition.In this study,by using transcriptome analysis method,genes transcriptional differences on physiological characteristics of MSR-1 cells those cultured under aerobic?high-oxygen?and micro-aerobic?low-oxygen?conditions were analyzed.Subsequently,a new protein encoded by MGMSRv2₂046 was focused to discuss its roles on cell growth,magnetosome formation and regulation of dissimilatory denitrification.Magnetosome synthesis ability of M gryphiswaldense MSR-1 in an auto-fermentor can be precisely controlled through strict control of dissolved oxygen concentration.The wild type of MSR-1 cell with and without magnetosome synthesized were harvested by cultivation in 7.5 L fermenter through controlling dissolved oxygen concentration of 0.5%and 30.0%,respectively.The transcriptome data of two kinds of cell showed that 77 genes were up-regulated and 95 genes were down-regulated significantly under low oxygen condition.These genes primarily involved in the categories of cell metabolism,transport,regulation,and unknown-function proteins.It was revealed some of physiological characteristics of MSR-1:the dissimilatory denitrification pathway was activated under low oxygen condition and it may be as supplemental energy available for magnetosome synthesis;the nutrient transport and physiological metabolism were slowed down under low oxygen situation,but this defect could be compensated by multi-copies of amino acid and carbon transport system liv and dct operons;the genes of magnetosome membrane proteins?Mam and Mms?were not directly regulated by oxygen signal,perhaps,they were constitutively expressed.A proposed regulatory network of differentially expressed genes reflected the complexity of physiological metabolism in MSR-1,and suggested that some yet-unknown functional proteins play important roles such as ferric iron uptake and transport during magnetosome synthesis.The transcriptome data provided a holistic view of the responses of MSR-1 cells to differing oxygen levels as well as evidence of that MSR-1 not only grow aerobically but also absorb large quantity of iron to synthesize magnetosome under micro-aerobic and anaerobic conditions.The transcriptome data showed that MGMSRv2₂046 was up-regulated under magnetosome formation condition,in order to discuss its roles on cell growth and magnetosome synthesis,MGMSRv2₂046 null mutant ?2046 was successfully constructed.Cellular physiological and biochemical detection results showed that deletion of MGMSRv2₂046 had barely effect on cell growth,however decreased the ability of iron absorption as well as cell magnetic response,and delayed magnetosome formation.In addition,the mutant turned to synthesize less,small diameter super paramagnetic particles confirmed by TEM?Transmission electronic microscope?observation and magnetic property detection.Comparison of main metabolic pathway gene transcription in WT and ?2046 by qPCR revealed that MGMSRv2₂046 probably involved in transcription of mms genes indirectly,regulated balance of cellular redox status,and promoted dissimilatory denitrification timely for energy generation,which boosted the formation and maturation of magneotosome.The dissimilatory denitrification was further focused on and transcriptional regulation of its genes were determined in WT and ?2046 providing different nitrogen source and oxygen concentration.The results revealed dissimilatory denitrification was induced by low oxygen signal,and exist of MGMSRv2₂046 rendered activation of this pathway at a relative higher oxygen level.Protein structure analysis suggested MGMSRv2₂046 and previous reported MGMSRv2₂946?MgFnr?belong to Crp/Fnr family protein but are not typical Crp or Fnr.C terminal of both proteins contain Fnr C terminal DNA binding conserved amino acid ETXSR.MGMSRv2₂046 protein were found to recognize 18 bp sequence?GCATTTGATTTTGGTCAA?of nap upstream containing Fnr-box by Footprinting.Combining EMSA and ChIP experiments suggested that MGMSRv2₂046 have ability to bind with upstream of itself and nap,nir,nor,nos operons.These results revealed that MGMSRv2₂046 break the limitation of traditional Fnr whose function is hypoxic dependent and enhance the threshold of oxygen level for activation of dissimilatory denitrification in MSR-1.Furthermore,proposed a working model to elucidate positive regulation and activation of both MGMSRv2₂046 and MGMSRv2₂946 on dissimilatory denitrification under micro-aerobic condition,and therefrom revise and supplement condition of nitrate respiratory coupling with energy generation.This study provides theoretical and experimental evidence of magnetosome formation under low nutrient and oxygen concentration,and expend new insight on regulatory pattern of dissimilatory denitrification in MSR-1.Meanwhile,these findings reveal the responsive strategy of MSR-1 when facing of aerobic,micro-aerobic,and anaerobic condition,deepen the knowledge of strong adaption of MTB,and ground the foundation for further investigation of biomineralization mechanism.