Biomedical subjects

P J Weisbeek

Publications and source records attributed to P J Weisbeek.

At least 19 recordsLinked to original sources

PubMed2001

Sucrose metabolism in plastids.

The question whether sucrose (Suc) is present inside plastids has been long debated. Low Suc levels were reported to be present inside isolated chloroplasts, but these were argued to be artifacts of the isolation procedures used. We have introduced Suc-metabolizing enzymes in plastids and our experiments suggest substantial Suc entry into plastids. The enzyme levansucrase from Bacillus subtilis efficiently synthesizes fructan from Suc. Targeting of this enzyme to the plastids of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) plants leads to high-level fructan accumulation in chloroplasts and amyloplasts, respectively. Moreover, introduction of this enzyme in amyloplasts leads to an altered starch structure. Expression of the yeast invertase in potato tuber amyloplasts results in an 80% reduction of total Suc content, showing efficient hydrolysis of Suc by the plastidic invertase. These observations suggest that Suc can enter plastids efficiently and they raise questions as to its function and metabolism in this organelle.

Chloroplasts↗

PubMed2000

The transit sequence of ferredoxin contains different domains for translocation across the outer and inner membrane of the chloroplast envelope.

Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross-linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence.

Amino Acid Sequence↗

PubMed2000

Low temperature sensing in tulip (Tulipa gesneriana L.) is mediated through an increased response to auxin.

Tulip (Tulipa gesneriana L.) is a bulbous plant species that requires a period of low temperature for proper growth and flowering. The mechanism of sensing the low temperature period is unknown. The study presented in this paper shows that the essential developmental change in tulip bulbs during cold treatment is an increase in sensitivity to the phytohormone auxin. This is demonstrated using a model system consisting of isolated internodes grown on tissue culture medium containing different combinations of the phytohormones auxin and gibberellin. Using mathematical modelling, equations taken from the field of enzyme kinetics were fitted through the data. By doing so it became apparent that longer periods of low temperature resulted in an increased maximum response at a lower auxin concentration. Besides the cold treatment, gibberellin also enhances the response to auxin in the internodes in this in vitro system. A working model describing the relationship between the cold requirement, gibberellin action and auxin sensitivity is put forward. Possible analogies with other cold-requiring processes such as vernalization and stratification, and the interaction of auxin and gibberellin in the stalk elongation process in other plant species are discussed.

Cold Temperature↗

PubMed1999

A bipartite sequence element associated with matrix/scaffold attachment regions.

We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.

Animals↗

PubMed1999

Mannose inhibits Arabidopsis germination via a hexokinase-mediated step.

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.

3-O-Methylglucose↗

PubMed1998

A novel signaling pathway controlling induced systemic resistance in Arabidopsis.

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid-nonaccumulating NahG plants. Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

Acetates↗

PubMed1997

Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis thaliana.

The Arabidopsis thaliana genome is currently being sequenced, eventually leading towards the unravelling of all potential genes. We wanted to gain more insight into the way this genome might be organized at the ultrastructural level. To this extent we identified matrix attachment regions demarking potential chromatin domains, in a 16 kb region around the plastocyanin gene. The region was cloned and sequenced revealing six genes in addition to the plastocyanin gene. Using an heterologous in vitro nuclear matrix binding assay, to search for evolutionary conserved matrix attachment regions (MARs), we identified three such MARs. These three MARs divide the region into two small chromatin domains of 5 kb, each containing two genes. Comparison of the sequence of the three MARs revealed a degenerated 21 bp sequence that is shared between these MARs and that is not found elsewhere in the region. A similar sequence element is also present in four other MARs of Arabidopsis.Therefore, this sequence may constitute a landmark for the position of MARs in the genome of this plant. In a genomic sequence database of Arabidopsis the 21 bp element is found approximately once every 10 kb. The compactness of the Arabidopsis genome could account for the high incidence of MARs and MRSs we observed.

Amino Acid Sequence↗

PubMed1997

Sucrose control of phytochrome A signaling in Arabidopsis.

The expression of the Arabidopsis plastocyanin (PC) gene is developmentally controlled and regulated by light. During seedling development, PC gene expression is transiently induced, and this induction can be repressed by sucrose. In transgenic seedlings carrying a PC promoter-luciferase fusion gene, the luciferase-induced in vivo luminescence was similarly repressed by sucrose. From a mutagenized population of such transgenic seedlings, we selected for mutant seedlings that displayed a high luminescence level when grown on a medium with 3% sucrose. This screening of mutants resulted in the isolation of several sucrose-uncoupled (sun) mutants showing reduced repression of luminescence by sucrose. Analysis of the sun mutants revealed that the accumulation of PC and chlorophyll a/b binding protein (CAB) mRNA was also sucrose uncoupled, although the extent of uncoupling varied. The effect of sucrose on far-red light high-irradiance responses was studied in wild-type, sun1, sun6, and sun7 seedlings. In wild-type seedlings, sucrose repressed the far-red light-induced cotyledon opening and inhibition of hypocotyl elongation. sun7 seedlings showed reduced repression of these responses. Sucrose also repressed the far-red light-induced block of greening in wild-type seedlings, and both sun6 and sun7 were affected in this response. The results provide evidence for a close interaction between sucrose and light signaling pathways. Moreover, the sun6 and sun7 mutants genetically identify separate branches of phytochrome A-dependent signal transduction pathways.

Arabidopsis↗

PubMed1995

Site-directed mutagenetic study on the role of negative patches on silene plastocyanin in the interactions with cytochrome f and photosystem I.

To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA.

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.

Bacterial Outer Membrane Proteins↗

PubMed1994

Expression of Anabaena PCC 7937 plastocyanin in Synechococcus PCC 7942 enhances photosynthetic electron transfer and alters the electron distribution between photosystem I and cytochrome-c oxidase.

The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.

Anabaena↗

PubMed1994

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

Amino Acid Sequence↗

PubMed1994

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

Bacillus subtilis↗

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

The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids.

The interaction of the precursor of the chloroplast protein ferredoxin with membrane lipids was studied in monolayer experiments in order to investigate the possible involvement of membrane lipids in the protein translocation process. The precursor efficiently and specifically inserts into a total lipid extract of its biological target the outer envelope membrane of chloroplasts. This interaction is mediated by the transit sequence as it can also be observed for the chemically prepared transit peptide of ferredoxin but neither for the ferredoxin apoprotein nor holoprotein. Interactions with the individual chloroplast lipids, monogalactosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, and phosphatidylglycerol are predominantly involved which corresponds to the results obtained for transit peptide fragments of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (van't Hof, R., Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol and phosphatidylcholine, suggesting that a loose lipid headgroup packing due to small lipid headgroups and/or electrostatic repulsions facilitates efficient insertion. The observed preferences for interaction of the precursor and transit peptide of ferredoxin for the chloroplast outer envelope membrane lipid extract and the presequence of cytochrome c oxidase subunit IV for the mitochondrial outer membrane lipid extract indicate that targeting sequence-lipid interactions contribute to organelle-specific protein targeting.

Amino Acid Sequence↗

PubMed1993

A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes.

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.

Binding, Competitive↗

PubMed1993

Identification and characterization of a siderophore regulatory gene (pfrA) of Pseudomonas putida WCS358: homology to the alginate regulatory gene algQ of Pseudomonas aeruginosa.

Genes encoding biosynthesis of pseudobactin 358 (a microbial iron transport agent) and its cognate outer membrane receptor protein, PupA, are transcribed only under iron limitation in plant growth-promoting Pseudomonas putida WCS358. Two cosmid clones were identified from a gene bank of WCS358 DNA which could independently and in an iron-dependent manner activate transcription from a WCS358 siderophore gene promoter in heterologous Pseudomonas strain A225. The functional region of one of the clones was localized by subcloning, transposon Tn3Gus mutagenesis, and DNA sequencing. Genomic transposon insertion mutants in the functional region lost the capacity to activate a siderophore gene promoter fusion transcriptionally; furthermore, these mutants no longer produced pseudobactin 358. The activating region consisted of a single gene designated pfrA (Pseudomonas ferric regulator). The pfrA gene codes for a single polypeptide, PfrA, of approximately 18 kDa, which has 58% identity to AlgQ (also known as AlgR2), a positive regulator involved in transcriptionally regulating alginate biosynthesis in Pseudomonas aeruginosa. Cross-complementation studies between the pfrA gene of P. putida and the algQ gene of P. aeruginosa revealed that pfrA can restore mucoidy (alginate production) in an algQ mutant and that algQ could poorly complement a pfrA genomic mutant. It is concluded that PfrA is involved in the positive regulation of siderophore biosynthetic genes in response to iron limitation; furthermore, pfrA and algQ appeared to be interchangeable between P. putida and P. aeruginosa.

Alginates↗

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↗