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Imrich Barák

Publications and source records attributed to Imrich Barák.

12 recordsLinked to original sources
PubMed2006

The thioredoxin system from Streptomyces coelicolor.

This paper describes the cloning, purification and characterization of thioredoxin (TrxA) and thioredoxin reductase (TrxR) from bacterial strain Streptomyces coelicolor . The genes of S. coelicolor encoding TrxA and TrxR were amplified by polymerase chain reaction, inserted into pET expression vector and used to overexpress these proteins in Escherichia coli . TrxA and TrxR were produced as the hexahistidine fusion proteins and were recovered from the cytoplasm as the soluble proteins. The activity of the purified recombinant proteins was demonstrated. The activity of TrxA was shown by efficient reduction of insulin and activity of NADPH-dependent TrxR was revealed by catalyses of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reduction reaction. The reduction reaction was fully dependent upon the presence of TrxA as intermediate electron carrier with the pH optimum 7.5 and the temperature optimum 29 degrees C. Km value of TrxR for TrxA was 0.217 +/- 0.02 microM.

Amino Acid Sequence↗
PubMed2005

Oligo-chip based detection of tick-borne bacteria.

We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.

Anaplasma↗
PubMed2005

From fundamental studies of sporulation to applied spore research.

Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by 'controversial' models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004.

Bacillus subtilis↗
PubMed2005

Where asymmetry in gene expression originates.

A general problem in developmental biology concerns the process by which cells of one type divide to give dissimilar daughter cells. Even though these daughter cells may be genetically identical, they can differ morphologically and physiologically and have different fates. As one of the simplest differentiation processes, Bacillus subtilis sporulation represents an excellent model system for studying cell differentiation. Several decades of study of this process have provided insight into cell cycle regulation and development. This review summarizes important advances in our understanding of asymmetric gene expression during spore formation with an emphasis on developmental stages that lead to asymmetric septum formation and especially to activation of the first compartment-specific sigma factor -sigma(F).

Bacillus subtilis↗
PubMed2003

The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.

The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.

Amino Acid Sequence↗
PubMed2003

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication.

This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium. Large and spacious PV with multiple non-replicating C. burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively. Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol. Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane. These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug. Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse. Collectively, these results demonstrate that C. burnetii protein synthesis, but not replication, is required for fusion between nascent C. burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV. However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.

Animals↗
PubMed2003

Fusogenicity of the Coxiella burnetii parasitophorous vacuole.

This study investigated whether C. burnetii protein synthesis and replication is required for maintenance of the fusogenic character of the Coxiella parasitophorous vacuole (PV). Vero cells were infected with C. burnetii, (Nine Mile strain in phase II) at a multiplicity of infection of approximately 10 and simultaneously treated with bacteriostatic concentrations of chloramphenicol or carbenicillin. At 96 h post-infection, cells were viewed by phase contrast microscopy for PV maturation. Mature, spacious PV containing multiple nonreplicating C. burnetii were clearly visible in infected Vero cells treated with the cell wall inhibitor carbenicillin. Conversely, mature, spacious PV did not form in cells treated with the protein synthesis inhibitor chloramphenicol. Rather, immunofluorescence microscopy revealed individual C. burnetii in small, tight PV scattered throughout the cytoplasm. Like mature PV, these PV localized with the lysosomal glycoprotein LAMP-1, but not the early endosome protein EEA.1. This result suggests that de novo C. burnetii protein synthesis, but not replication, is required for homotypic fusion and maturation of nascent C. burnetii PV. We next examined whether sustained C. burnetii protein synthesis is necessary for maintenance of PV fusogenicity. J774A.1 murine macrophage-like cells with mature C. burnetii PV were incubated with latex beads and the trafficking of beads to PV was quantified. Fusion of PV with bead-laden vacuoles was severely inhibited in cells treated with chloramphenicol. These results suggest that sustained C. burnetii protein synthesis is required for PV fusion with other vacuoles of the endocytic pathway.

Animals↗
PubMed2002

Bacillus subtilis division protein DivIVA - screen for stable oligomer state conditions.

The cell division protein DivIVA is predicted to be a coiled-coil, tropomyosin-like protein, that self-associates both in vivo and in vitro into oligomers of up to 10-12 monomers. A simple and quick screen for conditions supporting the stable oligomer structure has been developed revealing that DivIVA forms a homogeneous oligomer in the presence of PEGs (PEG 4 K or PEG 8K and PEG 1K).

Bacillus subtilis↗
PubMed2002

Dimer formation and transcription activation in the sporulation response regulator Spo0A.

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A.

Bacillus subtilis↗
PubMed2002

Two-vector assay as a tool for examining Spo0A gene transcription regulation.

We have modified an assay using two compatible vectors that coexist in Escherichia coli cells and applied it in the investigation of the transcriptional activity of Spo0A, a key regulator of sporulation in Bacillus subtilis. We have chosen the promoters of the Spo0A dependent genes, spoIIE and spoIIA, involved in sporulation, in order to study the transcription activity solely of the DNA binding domain of Spo0A. We have prepared the two-vector system so that one vector contained the cloned C-Spo0A under the control of an inducible promoter, and the second vector (the promoter probe vector), was composed of the Spo0A dependent spoIIE and spoIIA promoters. Using this two-vector system in E. coli, we proved that C-Spo0A is able to interact with the E. coli transcription apparatus, recognizes both promoters and activates transcription from these promoters.

Bacillus subtilis↗
PubMed2002

Oligomerization of the Bacillus subtilis division protein DivIVA.

DivIVA appears to be a mediator of inhibition by MinCD of division at the cell poles in Bacillus subtilis. Gel permeation and ultracentrifugation techniques were used to show self-association of DivIVA into a form consisting of 10-12 monomers in vitro. Western blot analysis of non-denaturating polyacrylamide gels confirms the presence of similar oligomers in B. subtilis cell lysates. These oligomers persist in a B. subtilis strain containing the divIVA1 mutation, in which proper vegetative septum positioning is abolished. In contrast, the divIVA2 mutation, which has a similar biological impact, appears to produce a protein with different oligomerization properties. The results of the present study suggest that oligomerization of DivIVA is important, but not sufficient for its function in the cell division process.

Bacillus subtilis↗