ABSTRACT
Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins , Escherichia coli Proteins , Escherichia coli/metabolism , Pseudomonas putida/metabolism , Receptors, Cell Surface/metabolism , Bacterial Outer Membrane Proteins/chemistry , Biological Transport, Active , Cloning, Molecular , Iron/metabolism , Receptors, Cell Surface/chemistry , Recombinant Fusion Proteins , Restriction Mapping , Siderophores/metabolism , Structure-Activity RelationshipABSTRACT
1. The effect of the peroxisome proliferators clofibrate and plasticizer on the activities of the first two enzymes involved in either phospholipid biosynthesis, i.e. dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone-phosphate synthase, were studied in rat liver homogenates and purified peroxisomes. 2. DHAP-AT in homogenates increased by 2 to 3-fold both in total and specific activity. However, the specific activity in purified peroxisomes showed no significant increase demonstrating for the first time that there is no specific induction of this enzyme that exceeds the induction of total peroxisomal protein. 3. Alkyldihydroxyacetone-phosphate synthase showed no significant increase in total and specific activity in homogenates and a slight decrease of its specific activity in purified peroxisomes was observed. 4. The total amount of plasmalogens did not increase upon proliferation and a slight decrease in the percentage plasmalogens in total phospholipids was observed. 5. Proliferation did not influence the phospholipid composition of the peroxisomal membrane.