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Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region.

ABSTRACT: We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in the P. aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in ptxS expression in both PAO1 and PAO::ptxS. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxS expression.

SUBMITTER: Swanson BL

PROVIDER: S-EPMC101966 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region.

Swanson B L BL Hamood A N AN

Journal of bacteriology 20000801 15

We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS ...[more]

PMID: 10894751

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Project description:Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.

Project description:UnlabelledMicrobial population polymorphisms are commonly observed in natural environments, including long-term infected hosts. However, the underlying processes promoting and stabilizing diversity are difficult to unravel and are not well understood. Here, we use chronic infection of cystic fibrosis airways by the opportunistic pathogen Pseudomonas aeruginosa as a system for investigating bacterial diversification processes during the course of infection. We analyze clonal bacterial isolates sampled during a 32-year period and map temporal and spatial variations in population diversity to different infection sites within the infected host. We show that the ancestral infecting strain diverged into distinct sublineages, each with their own functional and genomic signatures and rates of adaptation, immediately after initial colonization. The sublineages coexisted in the host for decades, suggesting rapid evolution of stable population polymorphisms. Critically, the observed generation and maintenance of population diversity was the result of partitioning of the sublineages into physically separated niches in the CF airway. The results reveal a complex within-host population structure not previously realized and provide evidence that the heterogeneity of the highly structured and complex host environment promotes the evolution and long-term stability of pathogen population diversity during infection.ImportanceWithin-host pathogen evolution and diversification during the course of chronic infections is of importance in relation to therapeutic intervention strategies, yet our understanding of these processes is limited. Here, we investigate intraclonal population diversity in P. aeruginosa during chronic airway infections in cystic fibrosis patients. We show the evolution of a diverse population structure immediately after initial colonization, with divergence into multiple distinct sublineages that coexisted for decades and occupied distinct niches. Our results suggest that the spatial heterogeneity in CF airways plays a major role in relation to the generation and maintenance of population diversity and emphasize that a single isolate in sputum may not represent the entire pathogen population in the infected individual. A more complete understanding of the evolution of distinct clonal variants and their distribution in different niches could have positive implications for efficient therapy.

Dataset Information

Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region.

Publications

Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region.

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