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Biomedical subjects

I Barák

Publications and source records attributed to I Barák.

At least 19 recordsLinked to original sources
PubMed2005

Thioredoxin - structural and functional complexity.

Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.

Animals↗
PubMed2004

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy.

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Bacillus subtilis↗
PubMed2004

Dimer-induced signal propagation in Spo0A.

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.

Bacillus subtilis↗
PubMed2001

Use of yeast two-hybrid system for detection of Bacillus subtilis FtsZ protein partners.

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with the ClaI restriction site shifted in the three possible reading frames in fusion with GAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ from Bacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of beta-galactosidase activity in yeast cells using 4-methylumbelliferyl beta-D-galactopyranoside as fluorogenic substrate.

Bacillus subtilis↗
PubMed2001

Bacillus subtilis locus encoding a killer protein and its antidote.

We have isolated mutations that block sporulation after formation of the polar septum in Bacillus subtilis. These mutations were mapped to the two genes of a new locus, spoIIS. Inactivation of the second gene, spoIISB, decreases sporulation efficiency by 4 orders of magnitude. Inactivation of the first gene, spoIISA, has no effect on sporulation but it fully restores sporulation of a spoIISB null mutant, indicating that SpoIISB is required only to counteract the negative effect of SpoIISA on sporulation. An internal promoter ensures the synthesis of an excess of SpoIISB over SpoIISA during exponential growth and sporulation. In the absence of SpoIISB, the sporulating cells show lethal damage of their envelope shortly after asymmetric septation, a defect that can be corrected by synthesizing SpoIISB only in the mother cell. However, forced synthesis of SpoIISA in exponentially growing cells or in the forespore leads to the same type of morphological damage and to cell death. In both cases protection against the killing effect of SpoIISA can be provided by simultaneous synthesis of SpoIISB. The spoIIS locus is unique to B. subtilis, and since it is completely dispensable for sporulation its physiological role remains elusive.

Bacillus subtilis↗
PubMed2000

A new mutation in spo0A with intragenic suppressors in the effector domain.

Spo0A is a two domain response regulator, a key protein in the initiation of sporulation of Bacillus subtilis. This protein controls a number of changes in gene expression that occur during the transition from stationary phase to the onset of sporulation. The phosphorylated form of Spo0A influences the transcription of a specific set of genes. In addition to others, it represses abrB and activates spoIIA and spoIIE transcription. Although the N-terminal phosphoacceptor domain is well characterised, there is limited information on the C-terminal, DNA-binding domain. Comparisons of Spo0A homologues from a number of Bacillus and Clostridium species show that the C-terminal domain contains three highly conserved regions. In this study, we have investigated the influence of spo0A mutations mapping within the C-terminal domain on transcription from the abrB, spoIIA and spoIIE promoters using lacZ fusions. Our results indicate that described mutations can be part of signalling between N- and C-terminal domains of the protein. Also, the increased expression observed from the spoIIE promoter in some Spo0A mutants might result from a stabilising function of these mutations on the transcriptional apparatus utilising sigma(A).

Amino Acid Sequence↗
PubMed2000

Domain swapping in the sporulation response regulator Spo0A.

Adaptive responses of micro-organisms, such as chemotaxis and sporulation, are governed by two-component systems consisting of sensor kinases, that interpret environmental signals, and response regulators which activate the appropriate physiological responses. Signal transduction via response regulator proteins is mediated through transient phosphorylation of aspartic acid residues. In Spo0A, the key regulator of development (sporulation) in Bacillus, phosphorylation of the N-terminal receiver domain (N-Spo0A) at aspartate-55 switches on the transcription activation functions residing in the C-terminal effector domain. Here we report the crystal structure of N-Spo0A from Bacillus stearothermophilus at 1.6 A spacing, revealing a dimer formed by an alpha-helix swap. Comparison of this structure with the recently described structure of phosphorylated N-Spo0A shows that dimer formation results from a cis-trans isomerization of the Lys106--Pro107 peptide bond. The quaternary reorganization is associated with alterations in the active site stereochemistry which may have implications for signalling. Remarkably, this 3-D domain swapped N-Spo0A dimer has an identical topology to a hypothetical CheY-like dimer, recently proposed as an intermediate in the evolution of the family of periplasmic substrate binding proteins.

Amino Acid Sequence↗
PubMed2000

The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography.

Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development. Its initiation is under the control of an expanded two-component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single alpha-helical domain comprising six alpha-helices in an unprecedented fold. The structure contains a helix-turn-helix as part of a three alpha-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the sigmaA-activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.

Amino Acid Sequence↗
PubMed1999

Phosphorylated aspartate in the structure of a response regulator protein.

Phosphorylation of aspartic acid residues is the hallmark of two- component signal transduction systems that orchestrate the adaptive responses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmental signals and a response regulator that activates the appropriate physiological response. Although structures of response regulators are known, little is understood about their activated phosphorylated forms, due to the intrinsic instability of the acid phosphate linkage. Here, we report the phosphorylated structure of the receiver/phosphoacceptor domain of Spo0A, the master regulator of sporulation, from Bacillus stearothermophilus. The phosphoryl group is covalently bonded to the invariant aspartate 55, and co-ordinated to a nearby divalent metal cation, with both species fulfilling their electrostatic potential through interactions with solvent water molecules, the protein main chain, and with side-chains of amino acid residues strongly conserved across the response regulator family. This is the first direct visualisation of a phosphoryl group covalently linked to an aspartic acid residue in any protein, with implications for signalling within the response regulator family.

Adaptation, Biological↗
PubMed1999

Green fluorescent protein as a detection marker for Coxiella burnetii transformation.

The molecular biological study of the obligate intracellular bacterium Coxiella burnetii is hampered because of the lack of an efficient DNA transformation system. We used expression of the green fluorescent protein (GFP) in addition to ampicillin resistance as a selection marker for detection of transformed C. burnetii cells. Fluorescent microscopy studies revealed that transformed C. burnetii cells can be detected easily inside the host cell line. A high level of GFP expression was reached with the strong Escherichia coli trc (trp/lac) promoter. The use of GFP not only provides a convenient marker for transformation of C. burnetii, but also allows detection of this obligate intracellular pathogen inside host eukaryotic cells. Possible applications for GFP in the study of host-pathogen interactions are discussed.

Ampicillin Resistance↗
PubMed1999

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2).

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.

Amino Acid Sequence↗
PubMed1999

Transient gene asymmetry during sporulation and establishment of cell specificity in Bacillus subtilis.

Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate. During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum. We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE. We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

Bacillus subtilis↗
PubMed1999

The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment.

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the sigma F factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of sigma F is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant sigma F activity, indicating that the septum requirement for activating sigma F can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

Bacillus subtilis↗
PubMed1999

Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A.

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2. 5 A spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 A spacing using synchrotron radiation.

Amino Acid Sequence↗
PubMed1998

MinCD proteins control the septation process during sporulation of Bacillus subtilis.

Mutation of the divIVB locus in Bacillus subtilis causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The divIVB locus contains five open reading frames (ORFs). The last two ORFs (minCD) are homologous to minC and minD of Escherichia coli but a minE homolog is lacking in B. subtilis. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. However, it has been proposed that MinCD has no essential role in sporulation septum formation. We have used electron microscopic studies to show septation events during sporulation in some minD strains. We have observed an unusually thin septum at the midcell position in minD and also in minD spoIIE71 mutant cells. Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site in minD cells. We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation of B. subtilis cells.

Adenosine Triphosphatases↗
PubMed1997

Disruption of a sigma factor gene, sigF, affects an intermediate stage of spore pigment production in Streptomyces aureofaciens.

The Streptomyces aureofaciens sigF gene encodes a sigma factor. By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained. This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation. Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores. The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation. The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.

Bacterial Proteins↗