| Siderophores,a type of low molecular weight iron chelator with high affinity of Fe(Ⅲ),are synthesized for iron intake by bacteria,fungi and other microorganisms or plants under the iron limiting condition.Pseudomonas,a genus of Gram-negative bacteria with wide distribution,great variety and strong adaptability,can produce a variety of siderophores with different structures.Recent studies have shown that the siderophores produced by Pseudomonas could not only help bacteria to absorb iron,but also were associated with the pathogenicity of the strains.Meanwhile,siderophores also have biological activity such as antibiotics,anti-tumor and anti-malaria agents,and metalloenzyme inhibitors.Pseudomonas donghuensis HYS is a strain from Wuhan East Lake,with high iron yield in the iron-limiting medium,was isolated and identified by our laboratory in earlier stage.Previous work proved that HYS produced two kinds of siderophores,pyoverdine and non-fluorescent siderophore.The non-fluorescent siderophore was identified as 7-hydroxytropolone(7-HT),secreted in large quantities by HYS.Our laboratory screened the genes related to 7-HT synthesis of HYS strain through transposon insertion mutation,and found that the genes affecting 7-HT synthesis were mainly in two gene clusters.Bioinformatics analysis found that cluster 1 with certain particularity only existed in six Pseudomonas strains,including HYS.Gene cluster 2 showed high homology with phenylacetic acid metabolic gene cluster of E.coli,so it was assumed that the cluster 2 was phenylacetic acid metabolic gene cluster.Based on previous work,this study will further verify the correlation between gene cluster 2 and 7-HT synthesis,as well as the function of paak1 and paak2 in mechanism of 7-HT biosynthesis.To further confirm the correlation between phenylacetic acid metabolic pathway and 7-HT biosynthesis in HYS,this study analyzed whether the related genes on gene cluster 2 could decompose aromatic compounds,and found that the gene deletion strains could not grow on the minimal medium with phenylacetic acid as a single carbon source.In addition,adding the phenylacetic acid to the iron-limited MKB medium could promote the production of 7-HT in these strains.It was confirmed that gene cluster 2 was involved in the metabolism of phenylacetic acid,and the phenylacetic acid metabolism took part in the synthesis of 7-HT.When analyzing the gene function of genes on cluster 1 and cluster 2,it was found that the protein sequence of paak1 on gene cluster 1 and paak2 on gene cluster 2 were highly homologous,both encoding phenylacetyl coenzyme A ligase,suggesting that these two gene products might be isozymes.The gene deletion strains ?paak2 and ?paak1?paak2 of HYS were constructed by homologous recombination method,and the cross-complement and single carbon source experiments were carried out on these strains to verify the functions of paak1 and paak2.The ?paak1 strain did not produce 7-HT,but could grow in the culture medium with phenylacetic acid as a single carbon source;the ?paak2 strain could produce 7-HT,but could not grow in the culture medium with phenylacetic acid as a single carbon source;the ?paak1?paak2 strain could neither produce 7-HT nor grow in the culture medium with phenylacetic acid as a single carbon source.It was found that the strains restored producing 7-HT through complement of paak1 or overexpression of paak2 in ?paak1 and ?paak1?paak2,and restored growth in the minimal medium with phenylacetic acid as a single carbon source through complement of paak2 or overexpression of paak1 in ?paak2 and ?paak1?paak2,but the growth of the ?paak1?paak2 strain complementing paak1 was slower and weaker than that of complementing paak2.In result,paak1 and paak2 in HYS are functionally complementary in the process of 7-HT biosynthesis,paak1 is mainly related to 7-HT biosynthesis,and paak2 mainly plays a major role in the catabolism of phenylacetic acid.Detecting the transcription levels of paak1 and paak2 found that paak1 and paak2 had expression difference in HYS,both of them were inhibited by high iron concentration.The expression level of paak1 was higher than that of paak2,and the inhibitory effect of high iron concentration on paak1 was stronger,which further confirmed the importance of paak1 in 7-HT biosynthesis.The transcription level of gene paak2 in the ?paak1 strain during the logarithmic phase(in this period the strain produces a large quantities of 7-HT)was about 10 times lower than that of the wild type.When the paak1 was supplemented in ?paak1 or when the strain was cultured for 24 h to a stable phase,the transcription level of paak2 was equal to that of the wild type.The transcription level of paak1 in the strain ?paak2 showed no significant difference from that of the wild type.Therefore,it was concluded that during the logarithmic phase of HYS,the 7-HT was produced in large quantities,gene paak1 was positively correlated with gene paak2.However,this phenomenon would disappear after the stable period,and the specific mechanism needs to be further analyzed.In the experiment,it was also found that ethanol could inhibit the synthesis of 7-HT by inhibiting the gene orf6-9 related to 7-HT biosynthesis,which proved that different carbon source metabolic systems would affect the biosynthesis of 7-HT.In summary,this study confirmed that gene cluster 2 was involved in the metabolism of phenylacetic acid,and the degradation pathway of phenylacetic acid was involved the pathway of 7-HT biosynthesis in HYS,which provided a direction for the study of the 7-HT biosynthesis pathway.According to this study,it was found that paak1 and paak2,which were located on two gene clusters and whose DNA sequences were far apart,were both related to the biosynthesis of 7-HT and showed expression difference.Paak1 is mainly related to the biosynthesis of 7-HT,and paak2 plays a major role in the decomposition of phenylacetic acid.Moreover,paak1 and paak2 are positively correlated in the period of which 7-HT is synthesized in large quantities in HYS.These results confirmed that paak1 and paak2 have complementary functions in HYS,but they are functionally different,which provides guidance for further study on the mechanism of 7-HT biosynthesis. |
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