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Archives of Microbiology (v.184, #2)
Time-course development of the Cd2+ hyper-accumulating phenotype in Euglena gracilis by César Avilés; M. Eugenia Torres-Márquez; David Mendoza-Cózatl; Rafael Moreno-Sánchez (pp. 83-92).
To determine the onset of the Cd2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg2+ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd2+ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl2, the Cd2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd2+ accumulated was similar for both control and Hg2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd2+ compartmentation into organelles.
Keywords: Euglena gracilis ; Cd2+ accumulation; Mercury; γ-glutamylcysteine; γ-glutamylcysteine synthetase; Glutathione; Phytochelatins
Time-course development of the Cd2+ hyper-accumulating phenotype in Euglena gracilis by César Avilés; M. Eugenia Torres-Márquez; David Mendoza-Cózatl; Rafael Moreno-Sánchez (pp. 83-92).
To determine the onset of the Cd2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg2+ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd2+ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress of the culture were analyzed. In cells exposed to 0.2 mM CdCl2, the Cd2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However, the stiochiometry of thiol-compounds/Cd2+ accumulated was similar for both control and Hg2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully inactivate Cd2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the Cd2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd2+ compartmentation into organelles.
Keywords: Euglena gracilis ; Cd2+ accumulation; Mercury; γ-glutamylcysteine; γ-glutamylcysteine synthetase; Glutathione; Phytochelatins
Nuclear translocation of the heterotrimeric CCAAT binding factor of Aspergillus oryzae is dependent on two redundant localising signals in a single subunit by Hideya Goda; Takashi Nagase; Shinjiro Tanoue; Junya Sugiyama; Stefan Steidl; André Tüncher; Tetsuo Kobayashi; Norihiro Tsukagoshi; Axel A. Brakhage; Masashi Kato (pp. 93-100).
The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a ‘piggy back mechanism’ in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.
Keywords: Aspergillus oryzae ; Hap complex; CCAAT-box; Nuclear localisation; Nuclear localisation signal
Nuclear translocation of the heterotrimeric CCAAT binding factor of Aspergillus oryzae is dependent on two redundant localising signals in a single subunit by Hideya Goda; Takashi Nagase; Shinjiro Tanoue; Junya Sugiyama; Stefan Steidl; André Tüncher; Tetsuo Kobayashi; Norihiro Tsukagoshi; Axel A. Brakhage; Masashi Kato (pp. 93-100).
The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a ‘piggy back mechanism’ in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.
Keywords: Aspergillus oryzae ; Hap complex; CCAAT-box; Nuclear localisation; Nuclear localisation signal
Mutations affecting transcription pausing in the Bacillus subtilis pyr operon by Hesheng Zhang; Casper Møller Jørgensen; Robert L. Switzer (pp. 101-107).
Pausing during transcription of the Bacillus subtilis pyr operon was proposed to play a role in its regulation by attenuation. Substitution mutations in the B. subtilis pyr DNA specifying the 3′-terminal nucleotides of the previously identified transcription pause sites substantially reduced pausing at these sites in vitro. This result confirms the general utility of this mutagenic strategy for studying transcriptional pausing. Pyrimidine-mediated repression in vivo of pyr-lacZ fusions containing some of these substitution mutations was substantially lower than those observed with the wild-type pyr-lacZ fusions. However, these defects in regulation were correlated with alterations in the stability of the terminator stem-loop specified by the attenuator, rather than with their effects on transcriptional pausing in vitro.
Keywords: pyrimidine biosynthesis; transcription pausing; attenuation; mutations; Bacillus subtilis
Mutations affecting transcription pausing in the Bacillus subtilis pyr operon by Hesheng Zhang; Casper Møller Jørgensen; Robert L. Switzer (pp. 101-107).
Pausing during transcription of the Bacillus subtilis pyr operon was proposed to play a role in its regulation by attenuation. Substitution mutations in the B. subtilis pyr DNA specifying the 3′-terminal nucleotides of the previously identified transcription pause sites substantially reduced pausing at these sites in vitro. This result confirms the general utility of this mutagenic strategy for studying transcriptional pausing. Pyrimidine-mediated repression in vivo of pyr-lacZ fusions containing some of these substitution mutations was substantially lower than those observed with the wild-type pyr-lacZ fusions. However, these defects in regulation were correlated with alterations in the stability of the terminator stem-loop specified by the attenuator, rather than with their effects on transcriptional pausing in vitro.
Keywords: pyrimidine biosynthesis; transcription pausing; attenuation; mutations; Bacillus subtilis
Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans by Atsuya Tabata; Isamu Yamamoto; Masahiro Matsuzaki; Toshio Satoh (pp. 108-116).
A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ß-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ß-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc 1 complex, did not affect the ß-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.
Keywords: Periplasmic nitrate reductase; napKEFDABC ; nap expression regulation; Denitrifying phototroph; Rhodobacter sphaeroides f. sp. denitrificans
Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans by Atsuya Tabata; Isamu Yamamoto; Masahiro Matsuzaki; Toshio Satoh (pp. 108-116).
A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ß-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ß-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc 1 complex, did not affect the ß-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.
Keywords: Periplasmic nitrate reductase; napKEFDABC ; nap expression regulation; Denitrifying phototroph; Rhodobacter sphaeroides f. sp. denitrificans
Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity by Alexis Dumoulin; Ulla Grauschopf; Markus Bischoff; Linda Thöny-Meyer; Brigitte Berger-Bächi (pp. 117-128).
DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of −131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.
Keywords: DsbA; Staphylococcus aureus ; Disulfide; Lipoprotein; Cysteine
Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity by Alexis Dumoulin; Ulla Grauschopf; Markus Bischoff; Linda Thöny-Meyer; Brigitte Berger-Bächi (pp. 117-128).
DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of −131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.
Keywords: DsbA; Staphylococcus aureus ; Disulfide; Lipoprotein; Cysteine
Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase by Thomas P. Werner; Nikolaus Amrhein; Florian M. Freimoser (pp. 129-136).
Inorganic polyphosphate (iPoP)—linear chains of up to hundreds of phosphate residues—is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP “overplus” were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP “overplus” was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth conditions.
Keywords: Inorganic polyphosphate; PolyP; Saccharomyces cerevisiae ; Yeast; “Overplus,” Growth phase; Diauxic shift; Polyphosphate quantification method
Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase by Thomas P. Werner; Nikolaus Amrhein; Florian M. Freimoser (pp. 129-136).
Inorganic polyphosphate (iPoP)—linear chains of up to hundreds of phosphate residues—is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP “overplus” were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP “overplus” was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth conditions.
Keywords: Inorganic polyphosphate; PolyP; Saccharomyces cerevisiae ; Yeast; “Overplus,” Growth phase; Diauxic shift; Polyphosphate quantification method
Expression of the UGA4 gene encoding the δ-aminolevulinic and γ-aminobutyric acids permease in Saccharomyces cerevisiae is controlled by amino acid-sensing systems by Mariana Bermudez Moretti; Ana Mercedes Perullini; Alcira Batlle; Susana Correa Garcia (pp. 137-140).
In yeasts, several sensing systems localized to the plasma membrane which transduce information regarding the availability and quality of nitrogen and carbon sources and work in parallel with the intracellular nutrient-sensing systems, regulate the expression and activity of proteins involved in nutrient uptake and utilization. The aim of this work was to establish whether the cellular signals triggered by amino acids modify the expression of the UGA4 gene which encodes the δ-aminolevulinic (ALA) and γ-aminobutyric (GABA) acids permease. In the present paper, we demonstrate that extracellular amino acids regulate UGA4 expression and that this effect seems to be mediated by the amino acid sensor complex SPS (SSY1, PTR3, SSY5).
Keywords: Saccharomyces cerevisiae ; Gene expression; UGA4 gene; Sensor systems
Expression of the UGA4 gene encoding the δ-aminolevulinic and γ-aminobutyric acids permease in Saccharomyces cerevisiae is controlled by amino acid-sensing systems by Mariana Bermudez Moretti; Ana Mercedes Perullini; Alcira Batlle; Susana Correa Garcia (pp. 137-140).
In yeasts, several sensing systems localized to the plasma membrane which transduce information regarding the availability and quality of nitrogen and carbon sources and work in parallel with the intracellular nutrient-sensing systems, regulate the expression and activity of proteins involved in nutrient uptake and utilization. The aim of this work was to establish whether the cellular signals triggered by amino acids modify the expression of the UGA4 gene which encodes the δ-aminolevulinic (ALA) and γ-aminobutyric (GABA) acids permease. In the present paper, we demonstrate that extracellular amino acids regulate UGA4 expression and that this effect seems to be mediated by the amino acid sensor complex SPS (SSY1, PTR3, SSY5).
Keywords: Saccharomyces cerevisiae ; Gene expression; UGA4 gene; Sensor systems