The Study On Mechanisms Of Biosynthesis And Regulation Of Siderophores In High-siderophore-yielding Pseudomonas Sp.HYS

Iron is absolutely crucial for almost all organisms to maintain normal life activities, as it functions as a cofactor for many metabolic enzymes mediating redox reactions and electron transfer. Although being the fourth most plentiful element in the Earth's crust, the iron available in the environment is insufficient, because Fe2+is easily oxidized and transforms to Fe(OH)3under physiological pH and aerobic conditions. Therefore in the long course of evolution, organisms have developed a range of ways to acquire iron, among which the most common way is the synthesis and secretion of siderophores. Bacteria are believed to usually produce more than one siderophore. The struggle for iron is also an important competition pathway between bacteria. Multiple siderophore systems, which are accurately regulated, are believed to enhance adaptability and confer an advantage on the producer.HYS, isolated from the water of East Lake of Wuhan, China, is a high-siderophore-yielding Pseudomonas strain. By the CAS liquid assay, the amount of siderophores produced by HYS is3-4times as high as that by some common Pseudomonas strains in the iron-limited MKB medium, implying the special iron uptake and metabolism system of HYS. To identify the siderophore-related genes in HYS, transposon mutagenesis was carried out. By screening19635transconjugants,60mutants that exhibited strongly different siderophore yields with wild type on CAS agar plates were obtained. Combining TAIL-PCR, arbitrarily primed PCR, and rescue cloning approaches, we obtained the transposon-flanking sequences and interrupted genes of all60mutants.22genes were involved in38mutants that can grow well on CAS agar plates. These genes could provide many materials and clues for further studies. To intensely study siderophores of HYS, we first analyzed the types of its siderophores. By deleting pvdA, whose defect abolishes the production of fluorescent siderophore pyoverdine, we found that HYS simultaneously secretes the fluorescent pyoverdine and the nonfluorescent siderophore, and that the nonfluorescent siderophore is the major type. Then the absorption spectra of the two types of siderophores in the culture supernatant were determined. The characteristic peaks of the nonfluorescent siderophore are at330nm and392nm, and the characteristic peak of fluorescent pyoverdine is at the previously reported405nm. In addition, the two types of siderophores had great differences in the production cycle, sensitivity to iron, and efficiency. Firstly, a large amount of the nonfluorescent siderophore accumulates from the exponential phase and reachs a peak before the stationary phase; while the fluorescent pyoverdine steadily accumulates until the stationary phase, but with a less yield. Secondly, the nonfluorescent siderophore tend to be produced under less drastically iron-limited conditions, but fluorescent pyoverdine under extremely iron-restricted conditions. Finally, the efficiency of pyoverdine appears to be greater than that of the nonfluorescent siderophore. The coexistence and complementation of the two different siderophores could enable HYS to adapt well to changing environments.After understanding that HYS produces two types of siderophores, we further chose the genes identified in our tansposon mutagenesis and explored the connection between them and siderophores. The two-componet system GacS/GacA and a special cluster composed of12open reading frames (ORFs) interested us. GacS/GacA two-componet system is a global signal transduction system and is highly conserved in Gram-negative bacteria. It has been demonstrated to be widely required for the production of many secondary metabolites, including extracellular enzymes, virulence factors, and biocontrol factors, and is responsible for many biological functions, such as pathogenicity and biocontrol. However, the relationship between GacS/GacA and siderophores, ubiquitous secondary metabolites, is still unclear. By constructing a series of derivative strains, we found that GacS/GacA positively controled the nonfluorescent siderophore production through the Gac/Rsm signal cascade; in contrast, inactivation of GacS/GacA led to upregulation of the fluorescent pyoverdine. This situation that GacS/GacA has different effects on two siderophores produced by one strain has not been previously reported. The differential effects may be connected with the expression of the two siderophores under different iron statuses. Through the different roles, HYS might be able to achieve the differentially expression and precise regulation of the two siderophores.In addition to the upstream regulatory system gacS/gacA, a special cluster which was screened with high frequency by transposon mutagenesis also had remarkable effects on siderophore production. This cluster is about14kb and is consist of12ORFs. Unexpectedly, such a string of genes was not found in other sequenced bacteria by sequence alignment. The predicted products in this cluster have not been reported to be directly related to siderophore biosynthesis. Besides, six of the12ORFs had high homology with bacteria of other genera, making this cluster even more special. We first performed gene deletions for nine ORFs whose transposon mutants have different siderophore yields compared to the wild type. The nonfluorescent siderophore production was abolished in seven deletion mutants, and slightly increased in one deletion mutant. These results confirmed that this cluster is responsible for nonfluorescent siderophore production, and we named it the nfs cluster. Three promoter sequences were used to construct the translational fusions to assess expression of the corresponding genes. We found that the three promoters were all repressed by high iron, suggesting that HYS could control nonfluorescent siderophore production by regulating expression of the nfs cluster under different iron conditions. In addition, expressions of the promoters of or/9-6and orf12were both positively controlled by gacA, and expression of orfl was also slightly affected by gacA. This result indicated that GacS/GacA system indeed controled nonfluorescent siderophore production through the nfs cluster.In conclusion, a series of siderophore-related genes obtained by transoposon mutagenesis provided lots of genetic information for the study on siderophores in HYS and other strains. This study revealed coexist and complementation of two different siderophores, and the different regulatory effects on them by GacS/GacA system in HYS, providing more insights into siderophore secretion strategy and regulatory mode of microbial siderophores. After finding that a novel cluster is related with the nonfluorescent siderophore and is controlled by the GacS/GacA system, we revealed a siderophore-related pathway in HYS, which provide more information for biosynthesis and regulation of siderophores.