Olga Troshina - Academia.edu (original) (raw)
Papers by Olga Troshina
Fems Microbiology Letters, 2002
The unicellular non-N2-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectiona... more The unicellular non-N2-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts.
Fems Microbiology Letters, 2001
Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homol... more Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned. The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL. Gene expression analyses show that these hyp genes are, like hupL, transcribed under N 2 -fixing but not under non-N 2 -fixing growth conditions. Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA. Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative 310 and 335 sequence elements and putative binding sites for the global nitrogen regulator NtcA. ß
Fems Microbiology Letters, 2002
The unicellular non-N 2 -fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectio... more The unicellular non-N 2 -fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts. ß : S 0 3 7 8 -1 0 9 7 ( 0 2 ) 0 0 8 9 4 -7
Current Microbiology, 1996
A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells ... more A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H2-uptake [2–7 μmoles of H2 · mg−1 (Chl a) · h−1] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H2 on in vivo H2-uptake was observed during the early stages of nitrogenase induction. However, exogenous H2 does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth.
Fems Microbiology Letters, 2001
Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homol... more Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned. The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL. Gene expression analyses show that these hyp genes are, like hupL, transcribed under N2-fixing but not under non-N2-fixing growth conditions. Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA. Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative −10 and −35 sequence elements and putative binding sites for the global nitrogen regulator NtcA.
Fems Microbiology Letters, 1997
Planta, 2004
Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strai... more Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H2 evolution. The uptake hydrogenase was identified in all N2-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N2-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N2-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H2 production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.
Current Microbiology, 2001
A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the... more A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coliΔargH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea · min−1· (mg protein)−1. Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer.
International Journal of Hydrogen Energy, 2002
Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. ... more Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. The basic and early applied research over the last 30 years has established the basis of present knowledge in the ÿeld and is a platform for future R& D directions. This work brie y surveys some of the progress made in the ÿeld of cyanobacterial hydrogen evolution during this time period. ?
Fems Microbiology Letters, 2006
The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine bi... more The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine biosynthesis, was demonstrated in the heterocystous cyanobacterium Nostoc PCC 73102 by measuring in vitro enzyme activity and by visualization of ASL in native protein gels. Activity staining of a native PAGE gel revealed one ASL-dependent band with a molecular mass of about 240 kDa. A colorimetric assay for ASL based on the measurement of urea produced from arginine in the presence of an excess of arginase was further used to analyze the cyanobacterial ASL. The in vitro ASL activity was highest in cells in exponential growth phase and decreased significantly during the stationary phase of growth, ranging from 4.4 to 0.8 nmol of product formed (mg protein)−1 min−1. Including arginine, citrulline or ornithine in the growth medium resulted in no significant change in the ASL activities, indicating that Nostoc PCC 73102 ASL is not regulated by metabolites of the ornithine cycle.
The present study was carried out in order to examine and characterize the bidirectional hydrogen... more The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.
International Journal of Hydrogen Energy, 2002
... References. 1. AJ Smith , Modes of cyanobacterial carbon metabolism. In: NG Carr and BA Whitt... more ... References. 1. AJ Smith , Modes of cyanobacterial carbon metabolism. In: NG Carr and BA Whitton, Editors, The biology of cyanobacteria, Blackwell Scientific Publications, Oxford (1982), pp. 4785. 2. LJ Stal and R. Mozelaar , Fermentation in cyanobacteria. ...
Fems Microbiology Letters, 2002
The unicellular non-N2-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectiona... more The unicellular non-N2-fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts.
Fems Microbiology Letters, 2001
Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homol... more Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned. The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL. Gene expression analyses show that these hyp genes are, like hupL, transcribed under N 2 -fixing but not under non-N 2 -fixing growth conditions. Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA. Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative 310 and 335 sequence elements and putative binding sites for the global nitrogen regulator NtcA. ß
Fems Microbiology Letters, 2002
The unicellular non-N 2 -fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectio... more The unicellular non-N 2 -fixing cyanobacterium Gloeocapsa alpicola CALU 743 contains a bidirectional hydrogenase. Parts of all structural genes, encoding the hydrogenase, were identified, cloned and sequenced. When comparing the sequences with analogous sequences from other cyanobacteria the highest similarity was observed with hox genes from Synechocystis sp. PCC 6803. The hydrogenase activity increased considerably when the cells were grown aerobically in a medium with limiting concentrations of nitrate. However, the relative abundances of hoxH and hoxY transcripts, detected by RT-PCR, did not change significantly, demonstrating that the increase in the activity of G. alpicola hydrogenase was not a result of the increase of the transcription. In contrast, in Anabaena variabilis the induction of a bidirectional hydrogenase activity correlated with the relative level of hoxH and hoxY transcripts. ß : S 0 3 7 8 -1 0 9 7 ( 0 2 ) 0 0 8 9 4 -7
Current Microbiology, 1996
A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells ... more A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H2-uptake [2–7 μmoles of H2 · mg−1 (Chl a) · h−1] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H2 on in vivo H2-uptake was observed during the early stages of nitrogenase induction. However, exogenous H2 does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth.
Fems Microbiology Letters, 2001
Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homol... more Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned. The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL. Gene expression analyses show that these hyp genes are, like hupL, transcribed under N2-fixing but not under non-N2-fixing growth conditions. Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA. Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative −10 and −35 sequence elements and putative binding sites for the global nitrogen regulator NtcA.
Fems Microbiology Letters, 1997
Planta, 2004
Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strai... more Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H2 evolution. The uptake hydrogenase was identified in all N2-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N2-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N2-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H2 production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.
Current Microbiology, 2001
A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the... more A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp. strain PCC 73102. The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da. Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms. In an Escherichia coliΔargH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine. Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea · min−1· (mg protein)−1. Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer.
International Journal of Hydrogen Energy, 2002
Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. ... more Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. The basic and early applied research over the last 30 years has established the basis of present knowledge in the ÿeld and is a platform for future R& D directions. This work brie y surveys some of the progress made in the ÿeld of cyanobacterial hydrogen evolution during this time period. ?
Fems Microbiology Letters, 2006
The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine bi... more The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine biosynthesis, was demonstrated in the heterocystous cyanobacterium Nostoc PCC 73102 by measuring in vitro enzyme activity and by visualization of ASL in native protein gels. Activity staining of a native PAGE gel revealed one ASL-dependent band with a molecular mass of about 240 kDa. A colorimetric assay for ASL based on the measurement of urea produced from arginine in the presence of an excess of arginase was further used to analyze the cyanobacterial ASL. The in vitro ASL activity was highest in cells in exponential growth phase and decreased significantly during the stationary phase of growth, ranging from 4.4 to 0.8 nmol of product formed (mg protein)−1 min−1. Including arginine, citrulline or ornithine in the growth medium resulted in no significant change in the ASL activities, indicating that Nostoc PCC 73102 ASL is not regulated by metabolites of the ornithine cycle.
The present study was carried out in order to examine and characterize the bidirectional hydrogen... more The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.
International Journal of Hydrogen Energy, 2002
... References. 1. AJ Smith , Modes of cyanobacterial carbon metabolism. In: NG Carr and BA Whitt... more ... References. 1. AJ Smith , Modes of cyanobacterial carbon metabolism. In: NG Carr and BA Whitton, Editors, The biology of cyanobacteria, Blackwell Scientific Publications, Oxford (1982), pp. 4785. 2. LJ Stal and R. Mozelaar , Fermentation in cyanobacteria. ...