The Biosynthetic Pathway Of The Pseudomonas Aeruginosa New QS Signal IQS

Quorum sensing(QS)is a widely conserved cell-to-cell communication mechanism which coordinates various bacterial community activities including virulence gene expression and biofilm formation.It is of vital importance to study bacterial quorum sensing mechanisms for design and developing new strategies to control and prevent bacterial infections.Pseudomonas aeruginosa employs a hierarchical quorum sensing(QS)network consisting of las,rhl,pqs and iqs regulatory elements to coordinate expression of bacterial virulence genes.Among them,IQS can be activated by phosphate-depletion stress,and is controlled by las and phoB systems.However,clinical isolates frequently contain loss-of-function mutations in the central las system.In 2013,Zhang's group have found a mechanism that could functionally substitute las,which led to identification of a novel QS signal IQS.Disruption of IQS biosynthesis paralyses the pqs and rhl QS systems and attenuates bacterial virulence.In this project,the research methods of molecular biology,genomics,analytical chemistry and biochemistry have been used to study the biosynthetic pathways of IQS.According to literature reports,four genes in the same operon(ambB?ambC ?ambD and ambE)are involved in the biosynthesis of IQS.First of all,we used HPLC and LC/LC-MS method to test the presence and changes of IQS related QS signals in the culture supernatants of the amb deletion mutants,strain ambBCDE-DH5 a and wild type strain PAO1,I found that amb mutations did not change the production of IQS,and heterologous expression strains(ambBCDE-DH5a)did not produce a compound with the peak of IQS(m/z 205),instead the antimicrobial compound AMB(m/z131.06)was detected.Bioinformatics analysis of the amb homologues unveiled a non-ribosomal peptide synthase(NRPS)gene cluster pch that encoding pyochelin biosynthesis.Earlier studies showed that pch cluster isinvolved in synthesis of an iron carrier.The pchA,B,C,D,E,F and G mutants were constructed,respectively,and the IQS production was determined using the supernatants of the corresponding mutants,The results showed that IQS production was paralyzed in the pch mutants,whereas in trans expression of the wild type pchE,in the pchE mutant restored IQS production.The results suggest that amb is not directly involved in the biosynthesis of IQS,instead pch genes are most likely involved in the synthesis of IQS.Bioinformatics prediction suggests that IQS synthetic substrates could be 1 molecule of salicylic acid(Sal)and 1 molecule of cysteine(Cys).We also confirmed that the precursor of IQS is the intermediate in the pyochelin biosynthetic pathway by addition of salicylic acid to the pchA,pchD,pchE mutants as a substrate and HPLC analysis.