The Study On The Toxicity-related Function Of σ Factor SigW And Anti-σ Factor RsiW System Involved In Pseudomonas Donghuensis HYS

Pseudomonas is widely distributed in nature and is a common type of environmental microorganisms.Pathogenic Pseudomonas has important research value in the pathogenic mechanism of bacteria.The Pseudomonas donghuensis HYS is a strain of Pseudomonas isolated from the donghu of our laboratory.The previous study in this laboratory found that its chronic lethal effect on Caenorhabditis elegans is stronger than PAO1 and PA14.A different chemotactic phenomenon is found in the process than previously reported,suggesting that the strain may have a different toxic mechanism and it is worth further exploration.In order to study the toxic mechanism of HYS strain against C.elegans,a mutant library of Pseudomonas donghuensis HYS transposon insertion was constructed,and68 mutant strains were obtained after primary screening and gene localization.Screening of transposon mutant library revealed that transmembrane protein anti-factor RsiW is involved in the regulation of bacterial toxicity.RsiW and extracellular sigma factor（ECF）SigW constitute the anti-sigma factor/sigma factor system.Sigma factor assists RNAP to specifically recognize promoter-initiated RNA synthesis.ECF sigma factor regulates bacterial toxicity by regulating the expression of virulence factor-related genes,which is one of the most important and common toxicity regulation mechanisms of pseudomonas.In order to clarify the relationship between the toxicity of HYS strain and the RsiW/SigW system,and to explore the toxicity regulation mechanism of HYS strain,the following work was carried out in this study.1.Validation of gene rsiW involved in HYS strain toxicity to C.elegans.Screening of the transposon insertion mutation library found that the 0201 7A mutant was less toxic to C.elegans than the wild-type HYS strain.A deletion mutant strain and complement strains constructed according to the genomic information of the transposon insertion site of the mutant strain.A chronic experiment was conducted to find that the LT₅₀ of C.elegans fed toΔrsiW was 5.053±0.143 d,which can prolong the C.elegans LT₅₀about 2 d relative to the wild type of HYS strains.The above results indicated that the gene rsiW in HYS strain was involved in its toxicity to C.elegans.2.The anti-factor RsiW and the factor SigW are involved in the toxicity of HYS strain to C.elegans.By analyzing the location of gene rsiW in the genome and combining with the prediction of bioinformatics,the co-transcription experiment was designed and it found that RNA polymerase subunit sigma-24 in front of gene rsiW were co-transcribed with rsiW.Yeast two-hybrid experiments revealed that the proteinσ²⁴ interacts with RsiW at the post-translational level,that is,σ²⁴ is SigW.Chronic lethal experiments of genes sig W and sig W-rsiW knockout and complement strains in HYS strains showed that the gene sig W is related to the toxicity of P.donghuensis HYS.Combining with real-time PCR analysis,it found that the system was involved in the toxicity of HYS in the form of differential expression of sig W relative to rsiW.The SigW/RsiW system is jointly involved in the toxicity of HYS to C.elegans and has dose effect,providing a basis for the subsequent study of the system’s role in the toxicity mechanism of HYS strains.3.Previous research in the laboratory has found that the Gac system,pantothenic acid synthesis pathway,Cbr/Crc system,and iron carrier synthesis pathway in HYS strains are related to toxicity.In order to explore the toxic regulatory pathway that HYS strain SigW,fluorescence quantitative experiments were designed to find that sig W is not regulated by Gac system at the transcriptional level,and sig W has no direct relation with key genes pan B,cbr A and pvd A in pantopantoic acid synthesis pathway,Cbr/Crc system and iron carrier synthesis pathway at the transcriptional level.This implies that SigW does not have a direct relationship with the known toxicity regulation system in HYS strains,and may work together through indirect relation or SigW with known four pathways for the parallel relationship,to provides the basis of background information support.To sum up,this study determined that the SigW/RsiW（factor/anti-factor system）is related to the toxicity of HYS strains,and mainly participates in the form of differential expression of sig W relative to rsiW.It found that SigW of HYS strain was not related to Gac system,pantothenic acid synthesis pathway,Cbr/Crc system and iron carrier synthesis pathway,which may indirectly regulate or participate in other virulence related pathways.This provides a new direction for studying the toxicity regulation mechanism of HYS strains,and lays a foundation for further research on the toxicity mechanism of HYS strains,and also provides ideas for the treatment of animal infections that may be caused by HYS strains in the future.