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T Börner

Publications and source records attributed to T Börner.

At least 19 recordsLinked to original sources
PubMed2001

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC.

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.

Cyanobacteria↗
PubMed2001

Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content.

We have constructed and analysed a cyanobacterial mutant that lacks the putative homologue of ycf37, the chloroplast open reading frame 37, which is conserved in different algae, but missing in the plastome of higher plants. In this report we show that Ycf37 of Synechocystis sp. PCC 6803 contains three tetratrico-peptide repeat (TPR) units resembling the structural organization of Ycf3, a protein that has been suggested to function as a chaperone during photosystem (PS) I complex formation. We demonstrate a light-activated transcript accumulation of this gene. Inactivation of ycf37 leads to a lower PSI/PSII ratio and a higher phycocyanin/chlorophyll ratio in Synechocystis cells. The observed alterations in the ycf37 mutants and the structural organization of the gene product suggest a functional role in PSI stability or assembly.

Amino Acid Sequence↗
PubMed2001

Molecular biology of peptide and polyketide biosynthesis in cyanobacteria.

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.

Cyanobacteria↗
PubMed2001

Characterization of the Cph1 holo-phytochrome from Synechocystis sp. PCC 6803.

The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6-tagged Cph1 was isolated from Synechocystis cells. The cph1-his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+-induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.

Bacterial Proteins↗
PubMed2001

Phosphorylation of proteins in the light-dependent signalling pathway of a filamentous cyanobacterium.

The genome of the filamentous cyanobacterium Calothrix sp. PCC7601 contains two genes, cphA and cphB, encoding proteins with similarity to plant phytochromes and bacterial histidine kinases. In vitro, CphA and CphB readily attach a tetrapyrrole chromophore to develop spectrally active holoproteins that are photointerconvertible between a red light-absorbing and a far-red light-absorbing form. Together with the putative response regulators, RcpA and RcpB, the putative histidine kinases, CphA and CphB, are suggested to constitute two two-component systems of light-dependent signal transduction. In this report, we demonstrate the kinase activity of both CphA and CphB. In vitro experiments carried out on the purified proteins show that CphA and CphB are autophosphorylated in the presence of ATP and that phospho-CphA is capable of efficient phosphotransfer to RcpA as is phospho-CphB towards RcpB. The autophosphorylation and the phosphorelay are dependent on light. Both activities are reduced under red light vs. far-red light irradiation. No phosphoryl transfer occurred between phospho-CphA and RcpB or between phospho-CphB and RcpA. The response regulators RcpA and RcpB can receive a phosphoryl moiety also from the small phospho-donor acetyl phosphate. The stability of the phosphorylated regulators is not affected by CphA and CphB or light.

Bacterial Proteins↗
PubMed2001

Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

Algal Proteins↗
PubMed2001

Effects of cell-bound microcystins on survival and feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.

Animals↗
PubMed2000

Phage T4-like intermediates of DNA replication and recombination in the mitochondria of the higher plant Chenopodium album (L.).

We have studied intermediates of the recombination and replication of chromosomal mitochondrial (mt) DNA prepared from suspension cultured cells of Chenopodium album (L.) by electron microscopy during the whole growth cycle. We identified several types of potential recombination and replication intermediates including rosette-like structures, as well as other branched and sigma-like molecules. The absolute and relative amounts of these structures changed dramatically during the growth cycle, indicating high dynamics in the structural organization of the mt genome. The rosette-like molecules had sizes of 2-5 genome units and were found to contain putative replication forks and 'Holliday'-junctions known from recombination intermediates. The high number of rosettes during the first days of culture, and their drastic reduction in the stationary growth stage, were found to be inversely related to changes in the quantity of linear molecules of 40-200 kb. This observation suggests that linear molecules participate in the formation of giant branched rosette-like structures. Most linear molecules were previously found to have at least one single-stranded end, which may allow recombinative invasion of other double-stranded molecules. Thus, recombination events may lead to the formation of more complex molecules and initiate replication similar to phage T4. We propose the coexistence of a recombination-dependent mode of replication with a presumably recombination-independent rolling-circle mode of replication in the mitochondria of C. album.

Bacteriophage T4↗
PubMed2000

Endochitinase gene-based phylogenetic analysis of Trichoderma.

DNA sequences of the single-copy gene coding for the 42 kDa endochitinase enzyme (EC 3.2.1.14) were used for phylogenetic analysis in Trichoderma. A set of 12 primers was developed and the entire gene was sequenced for 16 strains, and nucleotide and deduced amino acid sequences were compared to data from GenBank for additional Trichoderma strains. Analysis of the sequences revealed parsimony informative variation from 2.4 to 43.6% depending on the part of the gene (exons/introns) and the taxonomic level considered. Results are discussed in comparison to previous data from ITS-1 and ITS-2 rDNA sequencing and suggest the 42 kDa endochitinase gene as a potential molecular marker for reconstructing phylogenetic relationships in the genus Trichoderma at species level.

Chitinases↗
PubMed2000

Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system.

BACKGROUND: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Amino Acid Motifs↗
PubMed2000

One RNA polymerase serving two genomes.

The land plant Arabidopsis thaliana contains three closely related nuclear genes encoding phage-type RNA polymerases (RpoT;1, RpoT;2 and RpoT;3). The gene products of RpoT;1 and RpoT;3 have previously been shown to be imported into mitochondria and chloroplasts, respectively. Here we show that the transit peptide of RpoT;2 possesses dual targeting properties. Transient expression assays in tobacco protoplasts as well as stable transformation of Arabidopsis plants demonstrate efficient targeting of fusion peptides consisting of the N-terminus of RpoT;2 joined to green fluorescent protein to both organelles. Thus, RpoT;2 might be the first RNA polymerase shown to transcribe genes in two different genomes. RNA polymerase activity of recombinant RpoT;2 is uneffected by the inhibitor tagetin, qualifying the gene product of RpoT;2 as a phage-type polymerase.

Amino Acid Sequence↗
PubMed2000

Light and the transcriptional response of the microcystin biosynthesis gene cluster.

Microcystin, a hepatotoxin known to be the cause of animal and human deaths, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. The toxin is produced nonribosomally via a multifunctional enzyme complex, consisting of both peptide synthetase and polyketide synthase modules coded for by the mcy gene cluster. The recent identification of the mcy genes in the production of microcystin synthetase for the first time provides an avenue to study the regulation of microcystin production at a genetic level. In this study, M. aeruginosa PCC7806 was grown either under continuous light of various intensities or under low light with subsequent short-term exposure to different light intensities and qualities and various stress factors. RNase protection assays were employed to observe the level of mcyB and mcyD transcription under each condition. Both mcyB and mcyD transcript levels were increased under high light intensities and red light. Blue light and certain artificial stress factors (methylviologen and NaCl) led to reduced transcript amounts. There appeared to be two light thresholds, between dark and low light (16 micromol of photons m(-2) s(-1)), and medium (31 micromol of photons m(-2) s(-1)) and high light (68 micromol of photons m(-2) s(-1)), at which a significant increase in transcription occurred. Our findings show that the effect of light on microcystin synthetase production is due to light quality and is initiated at certain threshold intensities, which are not necessarily reflected by observed intracellular toxin bioactivity.

Cyanobacteria↗
PubMed1999

A gene family encoding glutathione peroxidase homologues in Hordeum vulgare (barley).

We have isolated and characterised three barley cDNAs encoding glutathione peroxidase (GPX) homologues, designated HVGPH1, HVGPH2 and HVGPH3. HVGPH1 may represent a cytosolic form of GPX. The structure of the HVGPH2 N-terminal domain is typical for a plastid transit peptide. A potential peroxisomal targeting sequence occurs near the N-terminus of HVGPH3. Transcript levels of HVGPH1 and HVGPH2 were increased in leaves undergoing stress. In contrast, HVGPH3 mRNA accumulation showed a negative response to stress. Our data indicate that the barley genome bears a small gene family encoding GPX homologues differing in their function and cellular localisation.

Amino Acid Sequence↗
PubMed1999

Comparative analysis of splicing of the complete set of chloroplast group II introns in three higher plant mutants.

The barley mutant albostrians and the maize mutants crs1 and crs2 are defective in the splicing of various plastid group II introns. By analysing tRNA precursors and several mRNAs not previously examined, the investigation of in vivo splicing defects in these mutants has been completed. The albostrians mutation causes the loss of plastid ribosomes resulting secondarily in a disruption of splicing of all subgroup IIA introns in the chloroplast. Thus MatK, the only putative chloroplast intron-specific maturase of higher plants, might have evolved to function in splicing of multiple introns. We show that in the case of tRNA-Ala(UGC)the first step of splicing is affected, as suggested by the absence of lariat molecules. Thus the plastid-encoded splicing factor lacking in albostrians must participate in the formation of the catalytically active structure. In contrast, a mutation in the nuclear gene crs1 prevents splicing of only one intron but causes specific additional effects as precursor transcripts for tRNA-Ile(GAU), tRNA-Ala(UGC), tRNA-Lys(UUU)and tRNA-Val(UAC), but not tRNA-Gly(UCC), have significantly enhanced steady-state levels in this mutant. Our data provide evidence for a variety of splicing factors and pathways in the chloroplast, some encoded by nuclear and some by chloroplast genes, and possibly for a dual function of some of these factors.

Base Sequence↗
PubMed1999

Cloning and characterization of three cDNAs encoding chloroplast RNA-binding proteins from barley (Hordeum vulgare L.): differential regulation of expression by light and plastid development.

Three cDNAs encoding chloroplast RNA-binding proteins (RNBPs) were identified in a screen of barley albostrians mutant expression libraries, and their binding and expression characteristics were determined. Two of these proteins, designated cp31AHv and cp31BHv, are closely related to group II, whereas the third, cp33Hv, is more similar to group III of the nuclear-encoded chloroplast RNBPs of dicot plants. Analysis of RNA from sections of primary leaves by Northern hybridization showed that the expression of these genes correlates with the stage of leaf development. The steady state transcript levels of the two genes encoding cp31BHv and cp33Hv, but not of the gene for cp31AHv, were positively affected by light. Moreover, a plastid factor is required for activation of cp31AHv transcription as revealed by the low level of cp31AHv mRNA in white leaves of the albostrians mutant and of seedlings treated with Norflurazon. Therefore, we propose the existence of a light-independent plastid-derived signal chain that regulates the expression of cp31AHv.

Amino Acid Sequence↗
PubMed1999

Organellar RNA polymerases of higher plants.

The nuclear genome of the model plant Arabidopsis thaliana contains a small gene family consisting of three genes encoding RNA polymerases of the single-subunit bacteriophage type. There is evidence that similar gene families also exist in other plants. Two of these RNA polymerases are putative mitochondrial enzymes, whereas the third one may represent the nuclear-encoded RNA polymerase (NEP) active in plastids. In addition, plastid genes are transcribed from another, entirely different multisubunit eubacterial-type RNA polymerase, the core subunits of which are encoded by plastid genes [plastid-encoded RNA polymerase (PEP)]. This core enzyme is complemented by one of several nuclear-encoded sigma-like factors. The development of photosynthetically active chloroplasts requires both PEP and NEP. Most NEP promoters show certain similarities to mitochondrial promoters in that they include the sequence motif 5'-YRTA-3' near the transcription initiation site. PEP promoters are similar to bacterial promoters of the -10/-35 sigma 70 type.

Amino Acid Sequence↗
PubMed1999

Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo.

The recent identification of phage-type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and in vitro import experiments. To determine the subcellular localization of the phage-type organellar RNA polymerases in plants, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenopodium album were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform A. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongreen plastids (leucoplasts) of root cells. By demonstrating targeting in plants, the data add substantial evidence for the phage-type RNA polymerases from C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.

Arabidopsis↗