Biomedical subjects

W Bitter

Publications and source records attributed to W Bitter.

25 records · Page 2Linked to original sources

PubMed1994

Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope.

The outer membrane protein PupB of Pseudomonas putida WCS358 facilitates transport of iron complexed to the siderophores pseudobactin BN8 and pseudobactin BN7 into the cell. Its synthesis is induced by the presence of these specific siderophores under iron limitation. The signal transduction pathway regulating siderophore-dependent expression of pupB was shown to consist of two regulatory proteins, PupI and PupR, and the PupB receptor itself. Mutational analysis of the regulatory genes suggested that PupI acts as a positive regulator of pupB transcription, whereas PupR modifies PupI activity dependent on the presence of pseudobactin BN8. PupI and PupR do not share homology with the classical bacterial two-component systems but display significant similarity to the FecI and FecR proteins of Escherichia coli involved in regulation of ferric dicitrate transport. The function of the PupB receptor in pupB regulation was studied by the use of chimeric receptor proteins composed of PupB and the ferric pseudobactin 358 receptor PupA. This experiment revealed that PupB is involved in the initiation of the signal transduction pathway, implying a so far unique role for an outer membrane protein in signal transduction.

Amino Acid Sequence↗

PubMed1994

Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.

Amino Acid Sequence↗

PubMed1994

Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain.

Bacterial Outer Membrane Proteins↗

PubMed1993

Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport.

Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes, for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.

Amino Acid Sequence↗

PubMed1991

The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein.

The initial step in the uptake of iron via ferric pseudobactin by the plant-growth-promoting Pseudomonas putida strain WCS358 is binding to a specific outer-membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N-terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB-dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55Fe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.

Amino Acid Sequence↗

PubMed1989

O-mannosylation of recombinant human insulin-like growth factor I (IGF-I) produced in Saccharomyces cerevisiae.

A glycosylated form of recombinant human insulin-like growth factor I (IGF-I) expressed in Saccharomyces cerevisiae was shown to contain mannose as the only carbohydrate constituent. All oligosaccharide chains of the glycoprotein could be released by mild alkaline treatment, and separated from the protein by gel-permeation chromatography on Bio-Gel P-2. The structures of these O-linked carbohydrate chains were determined by 500-MHz 1H-NMR spectroscopy, affording the disaccharide Man alpha 1-2Man as the major component and the tetrasaccharide Man alpha 1-3Man alpha 1-2Man alpha 1-2Man as a minor component. Reference oligosaccharides were prepared from mannoproteins released from the cell wall of S. cerevisiae X2180 (alpha-wild type). In addition to previously reported structures, ranging from mannose to mannotetraose, the pentasaccharide Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man was identified in the cell wall mannoprotein.

Carbohydrate Conformation↗

PubMed1989

The role of the positively charged N-terminus of the signal sequence of E. coli outer membrane protein PhoE in export.

Signal sequences of prokaryotic exported proteins have a dipolar character due to positively charged amino-acid residues at the N-terminus and to a preferentially negatively charged region around the cleavage site. The role of the two lysine residues at the N-terminus of the signal sequence of outer membrane protein PhoE of E. coli-K12 was investigated. Replacement of both of these residues by aspartic acid slightly affected the kinetics of protein translocation in vivo. This export defect, which was observed only when PhoE was overproduced, could not be suppressed by the prlA4 mutation, which has been shown to restore export defects caused by alterations in the hydrophobic core of the signal sequences of various exported proteins. In an in vitro translocation assay, the export defect was more pronounced. Replacement of both lysines by uncharged residues did not disturb the kinetics of protein export in vivo. In the in vitro assay, an extraordinarily efficient processing was detected upon incubation of this precursor with inverted cytoplasmic membrane vesicles. However, this efficient processing was not accompanied by more efficient translocation of the protein. We conclude that the positively charged residues at the N-terminus of the signal sequence are not essential for protein export, but contribute to the efficiency of the process.

Bacterial Outer Membrane Proteins↗