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Thomas Börner

Publications and source records attributed to Thomas Börner.

At least 19 recordsLinked to original sources
PubMed2006

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis.

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)-KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.

Computational Biology↗
PubMed2006

Transposons inactivate biosynthesis of the nonribosomal peptide microcystin in naturally occurring Planktothrix spp.

The filamentous cyanobacteria Planktothrix spp. occur in the temperate region of the Northern hemisphere. The red-pigmented Planktothrix rubescens bacteria occur in deep, physically stratified, and less eutrophic lakes. Planktothrix is a known producer of the toxic heptapeptide microcystin (MC), which is produced nonribosomally by a large enzyme complex consisting of peptide synthetases and polyketide synthases encoded by a total of nine genes (mcy genes). Planktothrix spp. differ in their cellular MC contents as well as the production of MC variants; however, the mechanisms favoring this diversity are not understood. Recently, the occurrence of Planktothrix strains containing all mcy genes but lacking MC has been reported. In this study, 29 such strains were analyzed to find out if mutations of the mcy genes lead to the inability to synthesize MC. Two deletions, spanning 400 bp (in mcyB; one strain) and 1,869 bp (in mcyHA; three strains), and three insertions (IS), spanning 1,429 bp (in mcyD; eight strains), 1,433 bp (in mcyEG; one strain), and 1,433 bp (in mcyA; one strain), were identified. Though found in different genes and different isolates and transcribed in opposite directions, IS were found to be identical and contained conserved domains assigned to transposable elements. Using mutation-specific primers, two insertions (in mcyD and mcyA) and one deletion (in mcyHA) were found regularly in populations of P. rubescens in different lakes. The results demonstrate for the first time that different mutations resulting in inactivation of MC synthesis do occur frequently and make up a stable proportion of the mcy gene pool in Planktothrix populations over several years.

Bacterial Proteins↗
PubMed2006

Relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and polynucleotide phosphorylase.

Plant mitochondrial genomes are extraordinarily large and complex compared to their animal counterparts, due to the presence of large noncoding regions. Multiple promoters are common for plant mitochondrial genes, and transcription exhibits little or no modulation. Mature functional RNAs are produced through various posttranscriptional processes, and control of RNA stability has a major impact on RNA abundance. This control involves polyadenylation which targets RNA for degradation by polynucleotide phosphorylase (PNPase). Here, we have analyzed polyadenylated RNA fragments from Arabidopsis plants down-regulated for PNPase (PNP- plants). Because of their polyadenylated status and the accumulation of the corresponding RNA in PNP- versus wild-type plants, these sequences represent mitochondrial RNA degradation tags. Analysis of these tags revealed that PNPase is involved in degrading rRNA and tRNA maturation by-products but also RNA transcribed from regions that are in some cases highly expressed although lacking known functional genes. Some of these transcripts, such as RNA containing chimeric open reading frames created by recombination or antisense RNA transcribed on the opposite strand of a known gene, may present potential detrimental effects to mitochondrial function. Taken together, our data show that the relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and PNPase.

Arabidopsis↗
PubMed2005

Development- and tissue-specific expression of the RpoT gene family of Arabidopsis encoding mitochondrial and plastid RNA polymerases.

Arabidopsis thaliana possesses three RpoT genes which encode three different phage-type RNA polymerases with yet unknown function in organelle transcription: RpoTm and RpoTp, imported into mitochondria and plastids, respectively, and RpoTmp, co-targeted into both organelles. Expression of the RpoT genes was analyzed by quantitative RT-PCR, histochemical beta-glucuronidase (GUS) assays and in situ hybridization. Transcripts of all three RpoT genes accumulated to very low amounts in all organs. Surprisingly, RT-PCR revealed their highest levels in flower tissues. RpoTm transcripts were the most abundant in all organs, except mature leaves, in which RpoTp transcripts showed the highest accumulation. In the developing seedling, RpoTm::GUS and RpoTmp::GUS expression precedes that of RpoTp::GUS, the latter showing up only 7 days after germination. The RpoTm and RpoTmp promoters expressed GUS mainly in meristematic and mitochondria-rich cells such as the distal part of the root and companion cells flanking the phloem, whereas RpoTp::GUS activity was found in green tissues as the parenchyme cells of young leaves, the primary cortex of the stem, and sepals of buds and young flowers. Sites of GUS expression coincided spatially with those of in situ hybridization. Our data demonstrate an overlapping expression pattern of RpoTm and RpoTmp, and a completely differing pattern of RpoTp expression. The results suggest that RpoTm and RpoTmp recognize different types of mitochondrial promoters. The plastid polymerase RpoTp might play a major role in green tissue, i.e. in chloroplast transcription, whilst the dual-targeted RpoTmp in plastids should function mainly in the transcription of genes in non-green types.

Arabidopsis↗
PubMed2005

Genetic contributions to the risk assessment of microcystin in the environment.

Of the known toxins produced by cyanobacteria, microcystins and nodularins are the most significant threat to human and animal health. Knock-out studies have confirmed that microcystins are produced nonribosomally by a multienzyme complex consisting of peptide synthetases, polyketide synthases, and tailoring enzymes. Gene clusters for microcystin biosynthesis have been identified and sequenced in the distantly related cyanobacterial genera Microcystis, Planktothrix, and Anabaena. Homologous genes have been detected in a nodularin-producing Nodularia strain. Subsequently, microcystin biosynthesis (mcy) genes have been used to establish molecular techniques for the detection of toxigenic cyanobacteria in laboratory and field studies. mcy genes of unknown origin can be assigned to the producing species. Techniques are currently being developed for the quantification of mcy genes in field populations. These initial genetic investigations pave the way for a molecular monitoring of microcystin- and nodularin-producing cyanobacteria and for studying the dynamics of toxic cyanobacteria in lakes. Furthermore, microcystin-deficient mutants have significantly increased our knowledge about the impact of the toxins on Microcystis-Daphnia interactions. The experience gained on microcystin biosynthesis genes will be valuable for a risk assessment of microcystin in the environment and for future water management and lake-restoration strategies.

Animals↗
PubMed2005

Red and far-red light alter the transcript profile in the cyanobacterium Synechocystis sp. PCC 6803: impact of cyanobacterial phytochromes.

Cyanobacteria possess genes encoding phytochrome-related proteins. We used a DNA microarray approach to evaluate the impact of the phytochromes Cph1 and Cph2 on red light (R)- and far-red light (FR)-dependent gene expression in the unicellular cyanobacterium Synechocystis sp. PCC 6803. In cells of wild-type and phytochrome mutants, one-fourth of all 3165 annotated putative protein encoding genes was light-responsive. R predominantly enhanced the expression of genes involved in transcription, translation, and photosynthesis, whereas FR upregulated the transcript level of genes known to be inducible by stress. The absence of Cph1 and/or Cph2 altered the light-dependent expression of about 20 genes. Hence, receptor(s) different from the two phytochromes are supposed to trigger the global R/FR alterations of the expression profile.

Bacterial Proteins↗
PubMed2005

Multiple promoters are a common feature of mitochondrial genes in Arabidopsis.

Mitochondrial genes in the plant Arabidopsis thaliana are transcribed by two phage-type RNA polymerases encoded in the nucleus. Little is known about cis-elements that are recognized by these enzymes and mediate the transcription of the Arabidopsis mitochondrial genome. Here, 30 transcription initiation sites of 12 mitochondrial genes and gene clusters have been determined using 5'-RACE and ribonuclease protection analysis of primary transcripts labelled in vitro by guanylyltransferase. A total of 9 out of 12 genes were found to possess multiple promoters, revealing for the first time that multiple promoters are a common feature of mitochondrial genes in a dicotyledonous plant. No differences in promoter utilization were observed between leaves and flowers, suggesting that promoter multiplicity reflects a relaxed promoter specificity rather than a regulatory role of promoter selection. Nearly half the identified transcription initiation sites displayed immediately upstream a CRTA core sequence, which was mostly seen within the previously described CRTAAGAGA promoter motif or a novel CGTATATAA promoter element. About as many promoters possessed an ATTA or RGTA core. Our data indicate that the majority of mitochondrial promoters in Arabidopsis deviate significantly from the nonanucleotide consensus derived earlier for dicot mitochondrial promoters.

Arabidopsis↗
PubMed2004

Overexpression of phage-type RNA polymerase RpoTp in tobacco demonstrates its role in chloroplast transcription by recognizing a distinct promoter type.

Plant cells possess three DNA-containing compartments, the nucleus, the mitochondria and the plastids. Accordingly, plastid gene regulation is fairly complex. Albeit plastids retained their own genome and prokaryotic-type gene expression system by a plastid-encoded RNA polymerase (PEP), they need a second nuclear-encoded plastid transcription activity, NEP. Candidate genes for putative NEP catalytic subunits have been cloned in Arabidopsis thaliana (AtRpoTp) and Nicotiana sylvestris (NsRpoTp). To provide evidence for RpoTp as a gene encoding a NEP catalytic subunit, we introduced the AtRpoTp and NsRpoTp cDNAs into the tobacco nucleus under the control of the strong constitutive CaMV 35S promoter. Analysis of transcription from NEP and PEP promoters in these transgenic plants using primer extension assays revealed enhanced transcription from typical type I NEP promoters as PatpB-289 in comparison with the wild type. These data provide direct evidence that RpoTp is a catalytic subunit of NEP and involved in recognition of a distinct subset of type I NEP promoters.

Arabidopsis↗
PubMed2004

The white barley mutant albostrians shows enhanced resistance to the biotroph Blumeria graminis f. sp. hordei.

We performed cytological and molecular analyses of the interaction between the biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei and white and green leaves of the barley albostrians mutant. The leaves have the same nuclear genotype but differ from each other in respect to plastid differentiation. White leaves showed enhanced penetration resistance to B. graminis f. sp. hordei, associated with higher epidermal H2O2 accumulation beneath the appressorial germ tubes and protein cross-linking in papillae. Very low basal salicylic acid content was found in white leaves, which further confirmed that H2O2 accumulation and penetration resistance in barley are independent of salicylic acid. Expression analysis of stress and defense-related genes, including such being involved in reactive oxygen species production and cell death regulation, revealed stronger constitutive or pathogen-induced transcript accumulation in white leaves. We discuss the data on the basis of the finding that white albostrians leaves exhibit a supersusceptible interaction phenotype with the hemibiotrophic fungus Bipolaris sorokiniana.

Ascomycota↗
PubMed2004

Chloroplast development affects expression of phage-type RNA polymerases in barley leaves.

We have identified the barley gene and cDNA encoding the plastid phage-type RNA polymerase (RNAP), nuclear-encoded plastid RNAP (RpoTp), and the nearly full-length cDNA of the mitochondrial RNAP, nuclear-encoded mitochondrial RNAP (RpoTm). RpoTp spans more than 9000 nt, consists of 19 exons and 18 introns, gives rise to a 3632-nt mRNA and is localized to the long arm of chromosome 1 (7H). The length of the deduced polypeptide is 948 residues. The mRNA levels of RpoTp and RpoTm were determined in roots and primary leaf sections of 7-day-old barley seedlings of the albostrians mutant, which were either phenotypically normal and exhibited a gradient of chloroplast development, or contained ribosome-deficient undifferentiated plastids. Transcript levels of RpoTp and RpoTm in almost all sections reached higher concentrations in plastid ribosome-deficient leaves than in the wild-type material, except in the most basal part of the leaf. These data indicate a role of plastid-to-nucleus signalling in the expression of the two RpoT genes. The mRNA levels of the plastid genes, beta-subunit of plastid-encoded RNAP (rpoB), proteolytic subunit of the Clp protease (clpP) and ribosomal protein Rpl2 (rpl2), all known to be transcribed by the nuclear-encoded RNAP (NEP), followed closely the pattern of RpoTp mRNA accumulation, strongly suggesting that RpoTp and NEP are identical. Transcripts of RpoTm and RpoTm-transcribed mitochondrial genes cytochrome oxidase subunit 2 (coxII) and ATPase subunit 9 (atp9) accumulated to the highest levels in the most basal parts of the leaf and declined considerably towards the leaf tip with a pronounced reduction in green versus white leaves. Our data revealed a marked influence of the developmental stage of the plastid on the expression and activity of organellar phage-type RNAPs and their target genes. Thus, interorganellar cross-talk in the regulated expression of nuclear-encoded plastid and mitochondrial RNAP genes might be a key element governing the concerted expression of genes located within plastids, mitochondria and the nucleus of the plant cell.

Amino Acid Sequence↗
PubMed2004

Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp.

To investigate the abundance of active and inactive microcystin genotypes in populations of the filamentous cyanobacterium Planktothrix spp., individual filaments were grown as clonal strains in the laboratory and analysed for microcystin synthetase (mcy) genes and microcystin. Twenty-three green-pigmented strains of P. agardhii originating mostly from shallow water bodies fell into two groups, those possessing mcyA and those lacking mcyA. In contrast, all of the 49 strains that were assigned to the red-pigmented P. rubescens contained mcyA. One strain of P. agardhii and eight strains of P. rubescens contained the total microcystin synthetase gene cluster but were found inactive in microcystin synthesis. To investigate the natural abundance of inactive mcy genotypes in P. rubescens individual filaments sampled from Lake Irrsee and Lake Mondsee (Austria) were analysed directly for the presence of mcyA and microcystin by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. All filaments assigned to P. rubescens contained mcyA. The proportion of inactive microcystin genotypes in populations with a low (Irrsee) or high density (Mondsee) of P. rubescens was 5% and 21%, each. The results of this study demonstrate that P. rubescens typically contain mcy genes whereas P. agardhii have a patchy distribution of mcy genes. In both species microcystin producers co-occur with non-microcystin producers due to the absence/inactivation of mcy genes.

Bacterial Proteins↗
PubMed2004

Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. Microcystin is synthesized nonribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55-kb microcystin synthetase (mcy) gene cluster. Also encoded within the mcy gene cluster is a putative ATP binding cassette (ABC) transporter, McyH. This study details the bioinformatic and mutational analyses of McyH and offers functional predictions for the hypothetical protein. The transporter is putatively comprised of two homodimers, each with an N-terminal hydrophobic domain and a C-terminal ATPase. Phylogenetically, McyH was found to cluster with members of the ABC-A1 subgroup of ABC ATPases, suggesting an export function for the protein. Two mcyH null mutant (DeltamcyH) strains were constructed by partial deletion of the mcyH gene. Microcystin production was completely absent in these strains. While the mcyH deletion had no apparent effect on the transcription of other mcy genes, the complete microcystin biosynthesis enzyme complex could not be detected in DeltamcyH mutant strains. Finally, expression levels of McyH in the wild type and in DeltamcyA, DeltamcyB, and DeltamcyH mutants were investigated by using immunoblotting with an anti-McyH antibody. Expression of McyH was found to be reduced in DeltamcyA and DeltamcyB mutants and completely absent in the DeltamcyH mutant. By virtue of its association with the mcy gene cluster and the bioinformatic and experimental data presented in this study, we predict that McyH functions as a microcystin exporter and is, in addition, intimately associated with the microcystin biosynthesis pathway.

ATP-Binding Cassette Transporters↗
PubMed2004

Involvement of cyanobacterial phytochromes in growth under different light qualities and quantities.

Inactivation of the genes for the cyanobacterial phytochromes cph1 and cph2 in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 affected the growth of the cells under certain light conditions. Differences in growth were detected by recording growth curves and in competition experiments. Mutation of cph1 and cph2 resulted in different effects. The cph1-mutant strains exhibited a reduced growth rate under far-red light (FRL), whereas the growth of the cph2-mutant strains was inhibited by red light (RL). The growth rate of a cph1- / cph2- double mutant was reduced under both RL and FRL. Furthermore, cph1-, cph2- as well as double-mutant strains showed impaired growth under high-light (HL) conditions. Acclimation of the photosynthetic apparatus of the mutants to RL, FRL and HL, as determined by pigment analysis, was similar to that of the wild type.

Bacterial Proteins↗
PubMed2003

Phylogenetic evidence for the early evolution of microcystin synthesis.

Cyanobacteria are a prolific source of secondary metabolites, including compounds with toxic and enzyme-inhibiting activities. Microcystins and nodularins are the end products of a secondary metabolic pathway comprised of mixed polyketide synthases and nonribosomal peptide synthetases. Both peptides are potent natural toxins produced by distantly related genera of cyanobacteria. Horizontal gene transfer is thought to play a role in the sporadic distribution of microcystin producers among cyanobacteria. Our phylogenetic analyses indicate a coevolution of housekeeping genes and microcystin synthetase genes for the entire evolutionary history of the toxin. Hence they do not corroborate horizontal transfer of genes for microcystin biosynthesis between the genera. The sporadic distribution of microcystin synthetase genes in modern cyanobacteria suggests that the ability to produce the toxin has been lost repeatedly in the more derived lineages of cyanobacteria. The data we present here strongly suggest that the genes encoding nodularin synthetase are recently derived from those encoding microcystin synthetase.

DNA-Directed RNA Polymerases↗
PubMed2003

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera.

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.

Anabaena↗
PubMed2003

The mcyF gene of the microcystin biosynthetic gene cluster from Microcystis aeruginosa encodes an aspartate racemase.

Microcystins are hepatotoxic, non-ribosomal peptides produced by several genera of freshwater cyanobacteria. Among other enzymic activities, in particular those of peptide synthetases and polyketide synthases, microcystin biosynthesis requires racemases that provide D-aspartate and D-glutamate. Here, we report on the cloning, expression and characterization of an open reading frame, mcyF, that is part of the mcy gene cluster involved in microcystin biosynthesis in the Microcystis aeruginosa strain PCC 7806. Conserved amino acid sequence motifs suggest a function of the McyF protein as an aspartate racemase. Heterologous expression of mcyF in the unicellular cyanobacterium Synechocystis PCC 6803 yielded an active His(6)-tagged protein that was purified to homogeneity by Ni(2+)-nitriloacetate affinity chromatography. The purified recombinant protein racemized in a pyridoxal-5'-phosphate-independent manner L-aspartate, but not L-glutamate. Furthermore, we have identified a putative glutamate racemase gene that is located outside the mcy gene cluster in the M. aeruginosa PCC 7806 genome. Whereas homologues of this glutamate racemase gene are present in all the Microcystis strains examined, mcyF could only be detected in microcystin-producing strains.

Amino Acid Isomerases↗
PubMed2003

Characterization of two Myb-like transcription factors binding to CAB promoters in wheat and barley.

The expression pattern and level of light-regulated genes are controlled by complex regulatory networks. Expression of genes encoding chlorophyll a/b-binding proteins of photosystem II is controlled by different photoreceptors and regulated primarily at the level of transcription. Light-dependent transcription of these genes is further modulated by the circadian system, affected by a developmental program and by a variety of environmental factors such as stress. Here we report the isolation of two Myb-like transcription factors from barley, HvMCB1 and HvMCB2, that bind specifically to defined regions of CAB promoters derived from wheat and barley. Deletion and mutation analysis of the wheat CAB1 promoter suggest that HvMCB1 and HvMCB2 are required for high-level but not for light- and circadian clock-regulated expression. Moreover, we demonstrate that the induction of HvMCB1 and HvMCB2 transcription is regulated differentially by environmental factors and plastid development. These observations indicate that HvMCB1 and HvMCB2, together with other, yet unknown regulatory factors, may mediate responsiveness of CAB gene transcription to a variety of environmental and developmental signals.

Amino Acid Sequence↗