Investigation On The Regulation Of Iron-responsive Genes In Pseudomonas Aeruginosa

Iron is an essential element for most organisms,including bacteria.Despite its abundance on earth,it is biologically unavailable in most environments.Iron limitation is the first line of defense for host against pathogens.On the other hand,reduced form of iron is highly toxic for most microorganisms.To obtain a balance in iron concentration,microbes evolved complex and smart heredity and biochemistry mechanisms to control iron uptake, metabolism and homeostasis.Bacterial quorum sensing is a density dependent global regulatory system.Iron regulation and quorum sensing intertwine in bacteria to form a complex regulation network.Pseudomonas aeruginosa is an important opportunistic pathogen that causes various diseases including chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections.It possesses an enormous genome and complex regulation mechanisms which control variety of cellular processes and bacterial behaviors including the production of virulence factors and pathogenicity during bacterial infection.This research firstly investigated the functions of two iron regulated genes,PA2572, encoding a probable two-component response regulator and PA3899,encoding probable sigrna-70 factor in the diferric dicitrate uptake pathway.The mutants of both genes were constructed in PAO1,and luxCDABE-based promoter-reporter fusions were also constructed to examine the expression of relevant genes in these mutants.The results indicate that PA2572 affected the expression of siderophore synthesis proteins PchDCBA and PvdF,and as well as the conserved hypothetical protein encoded by PA3849;PA3899 controlled the expression of pvdF and oprL.These observations suggest that both genes are related to siderophore-mediated iron uptake system.In PAO1,both genes did not affect the expressions of the quorum sensing(QS) system or the virulence factors.Instead,both genes were regulated by QS and iron affected both the expression of QS system and virulence factors and the formation of biofilm.In iron-rich culture medium,the biofilm formation of PAO1(â–³2572) and PAO1(â–³3899) were reduced compared to PAO1.The tol-oprL genes play important roles in maintaining outer membrane integrity, transmembrane transportation,and cell division in Gram-negative bacteria.In P.aeruginosa, the tol-oprL genes are organized uniquely in three operons,orf1-tolQRA,tolB and oprL-orf2, and are regulated by iron availability.TolQRA is similar to ExbBD-TonB complex and can imperfectly replace the function of the latter in iron transport system in P.aeruginosa.In this study,the promoters and expression profiles of the tol-oprL operons were characterized,the function of Orf1 investigated.Primer extensions were carried out by using both isotope-labelled and fluorescence labelled primers and the expression profiles were determined by using both lacZ and luxCDABE based transcriptional fusions.The results revealed two distinct promoters at the upstream region of tolQRA;one located in front of orf1 was constitutive while the other within the orf1 coding region was iron-regulated.Expression profiles indicate the tol-oprL operons were also down-regulated by the quorum-sensing systems during the late stage of growth.Unlike tolQ and tolA,a viable orf1 knockout strain was successfully constructed.The mutant exhibited altered cell and colony morphology, providing first evidence that Orf1 plays a non-essential role in the Tol-OprL complex in P. aeruginosa.