The Study Of Siderophore Biosynthesis Pathways In Shewanella Oneidensis

Iron is an essential element for almost all microorganisms.Although it is abundant in nature,it usually exists in an insoluble/highly insoluble compound state,such as ferric oxyhydroxide,which is almost unavailable for direct acquisition by organisms.The bioavailability of iron ions in the environment is about 10^-9-10^-18 mol/L,which is usually below the requirement for cells to grow optimally and to carry out vital physiological and metabolic processes.In order to survive,compete and reproduce,environmental microorganisms including bacteria have evolved an efficient iron acquisition mechanism:siderophore-mediated iron transport-increasing the bioavailability of iron ions,enabling cells to obtain iron in their natural habitats.Shewanella belong to the Gram-negative,?-proteobacteria,renowned for their extremely complex and diverse respiratory pathways,and therefore have great potential for application in bioremediation and bioenergy.The bacterial research model of this genus,S.oneidensis,has been always believed that it can only synthesize the sole siderophore?putrebactin?;but some Shewanella species/strains can synthesize three types of siderophores?putrebactin,avaroferrin,bisucaberin?.However,it is puzzling that all of these bacteria only own/use a set of highly conserved siderophore synthetases:PubABC.In this study,the question has been answered by dissecting the biosynthesis pathways of the siderophore substrate molecules.When the siderophore is secreted extracellularly,it binds to Fe³⁺to form a Fe³⁺-siderophore complex.The recognization and transport of Fe³⁺-siderophore complex through the outer membrane of the bacterial cell are mediated by the siderophore receptor.In S.oneidensis,the siderophore receptor PutA is responsible for responding to the Fe³⁺-putrebactin complex,the loss of PutA blocks the entry of the complex,leading to iron deficiency inside cells that in turn induces cells produce more putrebactin molecules,therefore,the putA mutant overexpresses putrebactin constitutively.In this work,we took advantage of the putA mutant to dissect biosynthesis pathways of siderophores and their substrate molecules in S.oneidensis.We identified three types of siderophores from the?putA supernatants:putrebactin,avaroferrin,bisucaberin.Among them,putrebactin is the main siderophore,the precursor is putrescine,the precursor of bisucaberin is cadaverine,and the precursors of avaroferrin are putrescine and cadaverine.The relative content of avaroferrin and bisucaberin increased by adding a certain amount of cadaverine in culture medium,which indicated that the cell substrate pool could change the ratio of siderophore producion.By using CAS assay and LC-MS,we demonstrated that putrescine was mainly synthesized from arginine through arginine decarboxylase?ADC pathway?,which was at odds with a previous finding that SpeF was the main putrescine biosynthesis enzyme.In addition,the loss of the putrescine synthesis pathway caused growth defects,indicating that ADC pathway was important for normal physiological activities of cells.Furthermore,we showed that putrescine biosynthesis was stable and not liable to the concentrations of iron and siderophores,and was not regulated by cellular iron homeostasis regulator Fur.And the expression level of spermidine synthase significantly decreased in?putA in which siderophore is produced in large quantities.As spermidine biosynthesis consumes putrescine,this result implies that in the low-iron environment,the majority of putrescine is used for siderophore synthesis,leading to a shortage for polyamine synthesis.For further validation,this study performed enzymatic analysis in vitro.Recombinant proteins of homologs of lysine and ornithine decarboxylases were expressed in Escherichia coli BL21?DE3?,including SO₁769,SO₁550,SpeA,SpeC,and SpeF,after purification,lysine or ornithine was used as the substrate for enzymatic reactions.Through HPLC analysis and determination of enzyme reaction constants,SpeC and SpeF were found to be bifunctional decarboxylases,both had lysine and ornithine decarboxylase activity.Among them,SpeC had the highest lysine decarboxylase activity,significantly exceeding SpeA and SpeF,indicating that SpeC plays an important role in converting lysine to cadaverine.Despite this,the major physiological role of SpeC and SpeF appeared still to decarboxylate ornithine given that their K_m for ornithine was lower than that for lysine.In addition,the expression of the speC gene was responsive to the concentrations of both siderophore and cadaverine.The promoter activity of the speC was significantly increased in?putA in which siderophore is produced in large quantities,and was significantly reduced in?pub in which no siderophores produced.And 2 mM exogenous cadaverine showed an inhibitory effect to speC,eliminated the up-regulation of speC resulting from the PutA loss.Overall,by identifying biosynthesis pathways of new siderophores and their substrate molecules,this thesis reveals the molecular mechanism by which S.oneidensis uses promiscuous activities of decarboxylase to synthesize diverse siderophores,providing a new insight into mechanisms for coordinated synthesis of multiple siderophores in bacteria.