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Biomedical subjects

G A van der Hofstad

Publications and source records attributed to G A van der Hofstad.

8 recordsLinked to original sources
PubMed1989

Cloning and characterization of a gene encoding an outer membrane protein required for siderophore-mediated uptake of Fe3+ in Pseudomonas putida WCS358.

In iron-limited environments plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. Ferric pseudobactin 358 is efficiently taken up by cells of WCS358 but not by cells of another rhizophere-colonizing strain, Pseudomonas fluorescens WCS374. A gene bank containing partial Sau3A DNA fragments from WCS358 was constructed in a derivative of the broad-host-range cosmid pLAFR1. By mobilization of this gene bank to strain WCS374 a cosmid clone, pMR, which made WCS374 competent for the utilization of pseudobactin 358 was identified. By subcloning of the 29.4-kilobase (kb) insert of pMR the essential genetic information was localized on a BglII fragment of 5.3 kb. Tn5 mutagenesis limited the responsible gene to a region of approximately 2.5 kb within this fragment. Since the gene encodes an outer membrane protein with a predicted molecular mass of 90,000 daltons, it probably functions as the receptor for ferric pseudobactin 358. The gene is flanked by pseudobactin 358 biosynthesis genes on both sides and is on a separate transcriptional unit. WCS374 cells carrying pMR derivatives with Tn5 insertions in the putative receptor gene did not produce the 90,000-dalton protein anymore and were unable to take up Fe3+ via pseudobactin 358. In WCS358 cells as well as in WCS374 cells the gene is expressed only under iron-limited conditions.

Bacterial Outer Membrane Proteins↗
PubMed1988

Siderophore-mediated uptake of Fe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizosphere microorganisms.

Under iron-limited conditions, Pseudomonas putida WCS358 produces a siderophore, pseudobactin 358, which is essential for the plant growth-stimulating ability of this strain. Cells of strain WCS358, provided that they have been grown under Fe3+ limitation, take up 55Fe3+ from the 55Fe3+-labeled pseudobactin 358 complex with Km and Vmax values of 0.23 microM and 0.14 nmol/mg of cell dry weight per min, respectively. Uptake experiments with cells treated with various metabolic inhibitors showed that this Fe3+ uptake process was dependent on the proton motive force. Furthermore, strain WCS358 was shown to be able to take up Fe3+ complexed to the siderophore of another plant-beneficial P. fluorescens strain, WCS374. The tested pathogenic rhizobacteria and rhizofungi were neither able to grow on Fe3+-deficient medium in the presence of pseudobactin 358 nor able to take up 55Fe3+ from 55Fe3+-pseudobactin 358. The same applies for three cyanide-producing Pseudomonas strains which are supposed to be representatives of the minor pathogens. These results indicate that the extraordinary ability of strain WCS358 to compete efficiently for Fe3+ is based on the fact that the pathogenic and deleterious rhizosphere microorganisms, in contrast to strain WCS358 itself, are not able to take up Fe3+ from Fe3+-pseudobactin 358 complexes.

Biological Transport, Active↗
PubMed1978

Initiation factor IF-3 and the binary complex between initiation factor IF-2 and formylmethionyl-tRNA are mutually exclusive on the 30-S ribosomal subunit.

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.

Escherichia coli↗
PubMed1978

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.

Escherichia coli↗
PubMed1977

The involvement of a complex between formylmethionyl-tRNA and initiation factor IF-2 in prokaryotic initiation.

A complex between initiation factor IF-2 and fMet-tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde. The IF-2 - FMet-tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di of triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF-2 - fMet-tRNA complex formation is not influenced by initiation factors IF-1 and IF-3. The binary complex can be bound to the 30-S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF-2 - fMet-tRNA complex and the nucleotide moiety to the 30-S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF-2 - fMet-tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate had no effect.

Aurintricarboxylic Acid↗
PubMed1976

Cooperative effects of initiation factors and fMet-tRNA in the formation of the 40-S initiation complex.

In this paper the mode of action of IF-1 in 40-S initiation complex formation was studied with MS2 RNA as messenger. Using initiation factors IF-2 and IF-3 labeled in vitro it appeared that IF-1 did not influence the binding of these factors in the absence of fMet-tRNA. However, in the presence of fMet-tRNA it was found that the enhancement of the fMet-tRNA binding by IF-1 was accompanied with an equimolar increase in binding of IF-2. Moreover, it appeared that also in absence of IF-1, fMet-tRNA binding is coupled with an equimolar enhancement of the IF-2 binding, which suggests the existence of a preribosomal complex between IF-2 and fMet-tRNA. The apparent Km values for both the binding of fMet-tRNA and IF-2 to 30-S subunits were determined and appeared to be equal, which makes a functioning of such a preribosomal complex in protein initiation very likely. The participation of GTP in this complex will be discussed. Functions of IF-1 in dissociation and recycling of IF-2, described by others, and the stimulation on the 30-S subunit level might well be explained as pleiotropic effects of one basic action of IF-1, i.e. a conformational change of 30-S subunits.

Binding Sites↗