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W C van Heeswijk

Publications and source records attributed to W C van Heeswijk.

15 recordsLinked to original sources
PubMed2000

Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein.

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

Adenine Nucleotides↗
PubMed2000

The Escherichia coli signal transducers PII (GlnB) and GlnK form heterotrimers in vivo: fine tuning the nitrogen signal cascade.

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)/NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII/GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII/GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.

Bacterial Proteins↗
PubMed1999

Studies on the roles of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control.

In Klebsiella pneumoniae, nitrogen fixation (nif) genes are regulated in response to fixed nitrogen and oxygen. The activity of the nif-specific transcriptional activator NifA is modulated by NifL, which mediates both oxygen and nitrogen control. The signal transduction protein GlnK is required to relieve the inhibitory effect of NifL on NifA that occurs when the intracellular N status is high and in a wild-type cell, the action of GlnK cannot be substituted by the structurally related protein PII. We have studied the modulation of NifA activity by NifL in an heterologous system in which the host organism is Escherichia coli. Using a DeltaglnB, DeltaglnK mutant, we have shown that the modulation of NifA activity by NifL is dependent on the concentration of GlnK in the cell and that when overproduced, PII can substitute for GlnK. Furthermore, our data suggest that PII can counteract the positive action of GlnK in relieving NifL-dependent inhibition of NifA activity. This negative effect of PII may be physiologically important in establishing repression of nif gene expression when the intracellular nitrogen status rises.

Bacterial Proteins↗
PubMed1998

GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition.

GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.

Adenosine Triphosphate↗
PubMed1998

Crystallization and preliminary X-ray analysis of Escherichia coli GlnK.

The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.

Bacterial Proteins↗
PubMed1997

The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.

Amino Acid Sequence↗
PubMed1996

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli.

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a sigma N (sigma 54) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.

Amino Acid Sequence↗
PubMed1995

An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade.

The PII protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a PII homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli PII. It contains the conserved tyrosine residue which is known to be the site of uridylylation in PII. E. coli is the first organism in which two different PII proteins have been detected.

Amino Acid Sequence↗
PubMed1993

The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli.

Regulation of glutamine-synthetase (GS) activity in enteric bacteria involves a complex cascade of events. In response to nitrogen limitation, a transferase catalyses the uridylylation of the PII protein, which in turn stimulates deadenylylation of GS. Deadenylylated GS is the more active form of the enzyme. Here we characterize in detail the genes from Escherichia coli encoding uridylyl-transferase (glnD), the PII protein (glnB), and adenylyl-transferase (glnE). glnD is transcribed from its own promoter, glnE is contranscribed with another gene, orfXE, whereas glnB is partly contranscribed with a gene encoding a homologue of the transcription activator NtrC. All three gln regulatory genes were constitutively expressed at a low level, i.e. their expression was independent of the nitrogen status and the RNA polymerase sigma factor sigma 54. We conclude that the functioning of the GS adenylylation cascade is regulated by modulation of the activities of uridylyl-transferase and adenylyl-transferase, rather than by changes in the expression of their genes.

Amino Acid Sequence↗
PubMed1990

Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation.

GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts.

Base Sequence↗
PubMed1988

Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast.

Two abundant factors, GFI and GFII which interact with the 5' flanking regions of nuclear genes coding for proteins of the mitochondrial respiratory chain have been identified. In one case (subunit VIII of QH2: cytochrome c oxidoreductase) the binding sites for both factors overlap completely and their binding is mutually exclusive. For the other 5' regions tested the GFI and GFII binding sites do not coincide. Interestingly, binding sites for GFI and GFII are also present in or at the 3' ends of the coding regions of two genes of the PHO gene family and in DNA elements important for optimal ARS and CEN function respectively. The sites recognized by GFI conform to the consensus RTCRNNNNNNACGNR, while those recognized by GFII contain the element RTCACGTG. We speculate that GFI and GFII may play a role in different cellular processes, dependent on the context of their binding sites and that one of these processes may be the coordination of the expression of genes involved in mitochondrial biogenesis with the progress of the cell cycle.

Base Sequence↗
PubMedDate not supplied

Multi-functional DNA proteins in yeast: the factors GFI and GFII are identical to the ARS-binding factor ABFI and the centromere-binding factor CPF1 respectively.

GFI and GFII are abundant DNA-binding proteins in the yeast Saccharomyces cerevisiae. Binding sites for GFI conform to the sequence 5'-RTCRYNNNNNACG-3'. This consensus can also accommodate the recognition sequence for the ARS1-binding factor ABFI. Results of retardation-competition assays, limited proteolysis experiments, molecular weight determinations based on denaturation-renaturation procedures and mobility shift assays of protein-DNA complexes formed in the presence of a monoclonal antibody raised against ABFI suggest strongly that GFI and ABFI are the same protein. Similarly, GFII appears to be identical to the centromere-binding protein CPF1 (alias CP1), since both proteins bind to the CDEI motif of yeast centromeres (5'-RTCACRTG-3') and cannot be detected in a cpf1 disruption mutant yeast strain. In addition, based on denaturation-renaturation studies, both factors appear to have molecular weights in the same range of 53-62 kDa.

Base Sequence↗
PubMedDate not supplied

Macromolecular intelligence in microorganisms.

Biochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of 'primitive' living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade in Escherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.

Cell Physiological Phenomena↗