Effects Of Exopolyphosphatase To Antimony(Ⅲ) Resistance In Pseudomonas Stutzeri TS44

As a prevalent toxic heavy metal, antimony (Sb) is widely distributed in the natural environment, such as in the soil, river, atmosphere, and has a wide range of industrial uses. Nowadays, with the increasingly development of the industrial utilization and the exploitation of the vast mineral resources, the antimony pollution becomes a serious problem. As a highly toxic heavy metal, trace antimony poisoning can cause serious body discomfort, and even organ failure and death. However, some organisms exist in nature could grown in niche with an extremely high concentration of antimony, thus, microorganisms play an important role in the antimony cycle. However, the reported researches related to Sb(Ⅲ)-resistant and Sb(Ⅲ)-oxidizing bacteria are very limited, especially for the molecular mechanisms. In this study, a highly Sb(Ⅲ) resistant and oxidizing strain P. stutzeri TS44 was used to investigate the mystery of the mechanisms in the Sb(Ⅲ)-resistant bacteria. Antimony resistant related genes were isolated, identified and functional analyzed.We used the Tn5 transposon insertion mutagenesis method to construct P. stutzeri TS44 mutant library. A total of 3000 transformants were selected and 3 mutants were obtained after repeated screening. We successfully isolated mutants flanking sequences: exopolyphosphatase(PPX) gene, cytochrome oxidase subunit coding gene, and orphan histidine protein kinase gene (QseB). As the previous literatures speculated that the ppx gene may be involved in pumping out copper cation, therefore, in this project, we mainly focused on exploring the potential function of ppx gene on bacterial antimony resistance. The ppx gene encoding exopolyphosphatase which could sequentially cut off the end of the high-energy phosphate bond of polyphosphates, releasing phosphorous ions and producing a large amount of energy to support bacterial growth. Gene knocking-out and complementation and qRT-PCR methods were further employed to explore the detail function of the ppx gene. We found out that the Sb(Ⅲ)-resistance level of ppx mutant (TS44Δppx) was decreased, while the TS44Appx complemented with ppx named TS44ΔppxC recovered to resistant phenotype as the wide type; the intracellular antimony concentration in TS44Δppx was three times higher than wild-type in early logarithmic phase; qRT-PCR results showed that ppx expression was induced by antimony; the intracellular polyphosphates content was decreased in wild-type strain when adding antimony. These results showed that the ppx gene has a significant contribution to pump out Sb(Ⅲ).The metabolism involved by ppx in P. stutzeri TS44 may be described according to the following pathway:Sb(Ⅲ) induced the ppx gene expression after been uptaken into cytoplasm by transport protein such as GlpF, and the enzyme activity of exopolyphosphatase was up-regulated at the same time, which could accelerate the hydrolysis of polyphosphates, releasing large amounts of phosphorus residues and producing energy for metabolism. The intracellular phosphate residues and extracellular hydrogen ion could form a transmembrane proton gradient channel, Sb(Ⅲ) was carried out by phosphorus residues, thereby reducing the toxicityto bacteria.In addition, in order to gain a more in-depth understanding of Sb(Ⅲ) efflux pathway, we knocked down several Resistance-Nodulation-Cell Division Family(RND) efflux genes with a relatively higher expression under the Sb(Ⅲ) inducing condition. However, the antimony-resistant phenotype did not change significantly in those mutants, indicating the Sb(Ⅲ) efflux process has not inhibited obviously. In this case, we conjecture that the efflux of Sb(Ⅲ) is a jointly metabolic process, which completed by multi-genes in a complex way. Further analysis is needed to verify the detail resistant mechanism.In this study, we clarified one of the antimony resistance mechanisms of strain TS44, and for the first time, reported that the ppx gene has a significant impact on resistance to Sb(III),which has an important reference value for subsequent researches on microbial antimony mechanism.