| Siderophores are ferric iron-chelating molecules with a high affinity for iron and a low molecular weight,200?2000 Da,and are synthesized and secreted by bacteria and fungi in response to iron deprivation.Pseudomonas,a genus of Gram-negative bacteria,contains a metabolically multifaceted group of organisms that occupy numerous ecological niches,with a strong adaptability.Pseudomonas produce a variety types of siderophores,siderophores not only help bacteria to despoil iron to overcome iron-deficiency,also are related to bacterial pathogenicity,and are of great significance to the research of Pseudomonas on biological control of diseases as well as medical science.Therefore,the developed and precisely regulated multiple siderophore systems in Pseudomonas are major factors for the strong adaptability of Pseudomonas to the environment,and confer a competitive advantage on the producer.Pseudomonas donghuensis HYS,isolated from the water of East Lake of Wuhan,China and classified as a newly specie,is a high-siderophore-yielding Pseudomonas strain,the amount of siderophore produced by HYS is 3-4 times of that produced by other common Pseudomonas strains in liquid MKB medium.HYS strain secretes two types of iron scavengers:pyoverdine and a nonfluorescent substance;the latter material was confirmed to be 7-hydroxytropolone(7-HT),which is present in substantial quantities in iron-deficient media in HYS.Our previous work screened out siderophore-related genes by using transposon insertion mutation.Gene orf6-9 was demonstrated to be the key gene of 7-HT biosynthesis,is regulated by extracellular iron density and the Gac/Rsm system.As a new type siderophore,7-HT also has toxicity,thus an elaborate regulation of 7-HT biosynthesis is indispensable.Combining transposon insertion mutation,gene knocking-out,real-time PCR and modified chrome azurol S(CAS)agar assays,genes involved in the synthesis of 7-HT were confirmed,the probable biosynthesis pathway of 7-HT was explored and a novel and complex regulation mechanism of 7-HT biosynthesis was uncovered in this work.1.Genes related to 7-HT biosynthesis were confirmed.To further verify the genetic basis of 7-HT,we used the pyoverdine-deficiency strain ?pvdA mutant as the parent strain,by screening 7811 transconjugants,15 mutants that exhibited strongly different siderophore yields with ?pvdA on CAS agar plates were obtained.Combining TAIL-PCR and arbitrarily primed PCR,9 genes were involved in all 15 mutants,of which 6 genes were in cluster 1.Combing library screening which used the wild-type HYS as the parent strain,cluster 1 and cluster 2 were determined to be siderophore-related clusters of P.donghuensis HYS.2.Through functional research and analysis of 7-HT-related genes,it was demonstrated that cluster 1 and cluster 2 are involved in 7-HT biosynthesis.The biosynthetic pathway of 7-HT was sketched out.Cluster 2 is a paa(Phenylacetic acid)cluster and shares a high similarity(48%-65%)with proteins encoded by genes in paa cluster of Escherichia.coli K12.The sole carbon utilization experiments showed that 7-HT-related insertion mutants of cluster 2 exhibited growth deficiency at different degrees when phenylacetic acid or its analogues was used as sole carbon source,indicating that cluster 2 is a paa cluster,which participates in 7-HT biosynthesis.It was reported that degradation pathway of phenylacetic acid takes part in tropolones biosynthesis,difunctional PaaZ catalytics both degradation of phenylacetic acid and synthesis of tropolones.ORF13 in cluster 2 was predicted to be a PaaZ,the overexpression of ORF13 depressed the yields of 7-HT in HYS strain when the site mutation of acetaldehyde dehydrogenase(ALDH)domain of ORF13 led to a restore of 7-HT in the AgacA mutant,demonstrating that ORF13 has dual-function and participates in 7-HT biosynthesis.The expression of ORF13 is regulated by iron density and Gac/Rsm system.A bioinformatic analysis indicated ten synthetases(orf2-orf11)and two regulators(orfl and orf12).Reverse transcription PCRs demonstrated that cluster 1 is composed of five operons,namely orf1,orf2-5,orf6-9,orf10-11 and orf12.Our previous study demonstrated that orf1,orf6-9 and orf12 are related to 7-HT production.The 7-HT production in deletion mutants of orf2-5 dropped partly,or was approximately equal to that in the wild type,indicating the influence of orf2-5 on 7-HT is weak or indirect.Deletion mutant of orf10 or orfll led to a 7-HT deficiency,indicating that orf10-11 upregulates 7-HT production.We found by using bioinformatic analysis that cluster 1 exists in only six strains including P.donghuensis HYS,suggesting the particularity of cluster 1.3.Two families of transcriptional regulators,LysR and TetR/AcrR,of cluster 1 are involved in the complex regulation mechanism of 7-HT biosynthesis.ORF1 is speculated to be an LysR family of transcriptional regulator,ORF12 displays homology with the TetR/AcrR family of transcriptional regulators.It was demonstrated in our study that iron density and Gac/Rsm system regulate the expression of ORF1 and ORF12 at different degrees,ORF1 and ORF12 are essential for the production of 7-HT through orf6-9.Our study showed that these two regulators have similar functions in the production of 7-HT through the same pathway.Therefore,we explored the relationship between ORF1 and ORF12 in 7-HT biosynthesis.Interestingly,we demonstrated that ORF1 is upstream of ORF12 in 7-HT biosynthesis,while regulators LysR and TetR are mainly arranged in parallel in bacterial metabolism.It may be a novel regulation model that two families of transcriptional regulators work serially,to carefully regulate the biosynthesis of metabolite such as 7-HT which have bioactivity.Meanwhile the overexpression of ORF12 depressed the transcription of orf9,and then led to a sharply drop on 7-HT production.Members of the TetR/AcrR family mainly act as repressors by forming homodimers or tetramers and binding to the operator of target DNAs.In this study,the repression of orf9 expression via the overexpression of ORF12 may also occur through the formation of dimers.In conclusion,the genetic basis of the novel siderophore 7-HT and functions of 7-HT-related genes were confirmed in this study.The probable 7-HT biosynthesis pathway was discussed.A complex and novel mechanism including Gac/Rsm system,ORF1 and ORF12 which belonged to two families of transcriptional regulators,of 7-HT biosynthesis was verified in P.donghuensis HYS,providing more information for the research of biosynthesis and regulation of siderophore as well as tropolone.This study elucidated the biological significance of elaborate regulation of 7-HT synthesis,enhancing the insight into strong adaptability and competitive advantage of Pseudomonas to complex and volatile bioenvironments. |
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