| As one of the basic metabolisms of microorganisms,iron metabolism maintains a variety of important physiological processes such as catalytic metabolic enzymes and DNA biosynthesis of cells.However,the limited iron concentration in the environment hinders the normal reproduction and niches adaptation of microorganisms,which not only affects the pathogenicity of various pathogenic bacteria,but also affects the antagonistic activity of biocontrol bacteria.In the course of long-term biological evolution,microorganisms have developed a variety of iron metabolism pathways,the most important of which is secreting high-affinity iron chelator-siderophore to help them obtain iron in low-iron environments.The evolution of new siderophores and the corresponding iron metabolic pathways is often originated from foreign genes(clusters)obtained by horizontal gene transfer(HGT).On the other hand,Ferric uptake regulator(Fur)widely presents in microorganisms as a central hub to coordinate the balance between different iron metabolism pathways.The preliminary work of this laboratory found that 4 Burkholderia seminalis strains S9,0901,DSM23518,and R456,respectively,played the role of environmental saprophytic bacteria,plant and human pathogens and antagonism biocontrol,and these specific roles are largely due to the specific niches’ adaption of each strain in the longterm evolution process.Through multidimensional omics and phenotype analysis,we found that adaption was due to the epigenetic means such as HGT,differential expression of sites and methylation to a large extent which enriched their respective iron metabolism pathways,especially the strain R456 that could antagonize fungus by secreting the antifungal substance c FP(cyclic dipeptide)which has an HGT-driven and iron-related gene cluster expressing in specific site in the genome.Therefore,with strain R456 as the object,in-depth research on the new iron metabolism derived from HGT and its relationship with Fur will not only greatly enrich the microbial iron metabolism network,but also help to make better use of beneficial microorganisms such as R456.Through research,this article has achieved the following main results:Firstly,in this study,a new nonribosomal peptide synthetases(NRPSs)gene cluster was identified in B.seminalis R456,which was transferred from the algae.This is also the first gene cluster found in the world transferred from algae to bacteria.Genomic analysis found that the strain had both two biosynthetic gene cluster of siderophore ornibactin and pyochelin,as well as this horizontal transfer gene cluster NRPS-R456.The NRPS gene cluster may participate in iron metabolism pathways mainly through affecting iron metabolism related phenotypes such as siderophore synthesis,growth under iron stress,biofilm formation and motility.Through high performance liquid chromatography-mass spectrometry,the strain was identified to synthesize ornibactin and related homologues and two pyochelin isomers under iron deficient conditions.Further,the two types of siderophores were separated and purified by preparative chromatography and nickel ion chromatography columns and their activities were identified.Combining the mass spectrometry data with the results of the bioinformatics analysis of the gene cluster,the gene cluster was predicted to synthesize a novel polypeptide with siderophore activity;At the same time,the deletion of the core gene of this gene cluster significantly reduced the synthesis of ornibatcin and pyochelin.Fluorescence quantitative PCR also showed that the expression of these two siderophore direct regulatory factors in the mutant was significantly down-regulated.Secondly,this study identified the interaction between the aforementioned HGT gene cluster NRPS-R456 and Fur,a traditional global regulator of iron metabolism in B.seminalis R456.First of all,this study showed that under iron-rich conditions,the absence of Fur significantly increased intracellular iron content,decreased catalase activity,and significantly weakened hydrogen peroxide tolerance,confirming the regulation of Fur on iron balance and avoiding excessive iron absorption under ironrich conditions;secondly,in order to clarify which proteins Fur exerts in its regulatory function,we found the interaction proteins through Fur protein prokaryotic expression,Fur protein antibody preparation and co-precipitation of immune proteins,and verify the interaction between Fur and proteins caught by Fur antibodies by bacterial two hybrid.Mass spectrometry identified proteins that interacting with Fur including ribosomal protein S1(Rps A),heat shock protein(Gro EL),transcription termination protein(Nus A),aldehyde dehydrogenase(Adh E),aspartate ligase(Asp S),phosphoenolpyruvate carboxykinase(Pck G)and heat shock protein(Dna K);finally,fluorescent quantitative PCR identified NRPS-R456 cluster and Fur both downregulated expression in each other’s mutants,and bioinformatics analysis found the existence of recognition sequence bound by Fur dimer in NRPS-R456,and yeast single hybrid show that the Fur protein could bind to the recognition sequence under iron-rich conditions,thereby inhibiting the expression of the gene cluster.These results clearly show that the horizontal transfer gene cluster NRPS-R456 may cooperate with the Fur protein in regulating iron metabolism in bacteria.Finally,in this paper,the Tn5 transposon mutation method was used to identify the relationship between iron metabolism controlled by NRPS-R456 and the antagonistic ability of bacteria.Firstly,a mutant library containing 997 R456 transposon mutants was constructed by random insertion of Tn5;then through iron deficiency medium screening,3 mutants with obvious growth restriction in iron deficiency environment were obtained,and through confrontation culture,10 mutants were screened with significantly weakened ability to antagonize rice sheath blight fungus,and another mutant with both weakened phenotypes;finally,the inserted gene was identified by reverse PCR,and it was found that mutant with double weakened phenotypes was inserted into the NRPS-R456(R456＿2057),three mutants with restricted growth in iron-deficient environment in which the transposons were inserted into glycine synthase(Pur L)and aspartate aminotransferase(Asp B)involved in the purine pathway,ironsulfur cluster protein;of the 10 mutants with weakened antagonists,8 mutants in which transposons were respectively inserted into glycosyltransferase,histone H1,and 50 S ribosomal protein L11 methyltransferase,membrane integrity-related transport protein(Pqi C),t RNA uridine synthase(Mnm G),sulfate transport protein(Yei H),catalase,sulfate adenosine transferase(Cys D),and the remaining 3 mutants in which transposons were inserted into the plasmid,of which 2 mutant strains Tn5 transposon inserted into the same hypothetical protein(R456＿6210),1 mutant strain transposon inserted into another hypothetical protein(R456＿6327).In summary,this paper identified an NRPS-R456 gene cluster horizontally transferred from algae in the antagonistic bacteria B.seminalis R456,which not only has a global iron metabolism regulation function,but also interacts with the traditional iron metabolism global regulation factor Fur.Random insertion mutation analysis showed that the bacterial specific antagonistic ability may also be largely related to the iron metabolism responsible for this NRPS-R456 gene cluster.This research results not only highlight the role of horizontal transfer in the evolution of bacterial iron metabolism,but also emphasize the importance of iron metabolism evolution in the bacteria adaptation to specific ecological sites resulting in specific biological functions. |
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