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F Meinhardt

Publications and source records attributed to F Meinhardt.

At least 19 recordsLinked to original sources
PubMed1999

The URA5 gene encoding orotate-phosphoribosyl transferase of the yeast Kluyveromyces lactis: cloning, sequencing and use as a selectable marker.

A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5 gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5 genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a 1.9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and an incomplete open reading frame strikingly similar to SEC65 of Saccharomyces cerevisiae and other yeasts, in which the gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 were selected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactis URA5 as the selectable marker, thereby proving functionality of the cloned gene.

Amino Acid Sequence↗
PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗
PubMed1999

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.

Blotting, Western↗
PubMed1999

Regulation of beta-galactosidase expression in Bacillus megaterium DSM319 by a XylS/AraC-type transcriptional activator.

The beta-galactosidase-encoding bgaM gene of Bacillus megaterium DSM319 and the divergently orientated bgaR operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of bgaM; a potential regulator binding site overlaps the bgaM promoter. A mechanism for regulation of beta-galactosidase expression in B. megaterium is proposed.

Bacillus megaterium↗
PubMed1998

Kluyveromyces lactis killer plasmid pGKL2: evidence for a viral-like capping enzyme encoded by ORF3.

ORF3 of the cytoplasmic linear plasmid pGKL2 was disrupted in vivo by integration of a selectable marker. Long-term cultivation of transformants carrying hybrid plasmids with a disrupted ORF3 under selective pressure did not deprive strains of the native counterpart, thereby proving its essentiality for pGKL2 replication and maintenance. The predicted ORF3 polypeptide was found to contain conserved motifs acquainted with mRNA-capping enzymes in the required order, just as in cytoplasmic viruses; new conserved motifs were also identified.

Acid Anhydride Hydrolases↗
PubMed1997

Microbial linear plasmids.

While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well. They are now known to be common genetic elements in both, pro- and eukaryotes. Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini. Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani. Plasmids with 5' attached proteins constitute the largest group. In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g. formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source. In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions. In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains. In Neurospora spp. and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids. This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology.

Bacteria↗
PubMed1997

ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo.

ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa. RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically. Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids. Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability. The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli. Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa. This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts.

Blotting, Northern↗
PubMed1997

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.

Amino Acid Sequence↗
PubMed1996

Kluyveromyces lactis killer system: analysis of cytoplasmic promoters of the linear plasmids.

All of the 14 genes encoded by the cytoplasmic linear killer plasmids of Kluyveromyces lactis are preceded by upstream conserved sequences (UCSs), cis-acting elements involved in plasmid gene transcription. Using the bacterial glucose-dehydrogenase gene as a reporter, expression driven by seven cytoplasmic promoters was determined. The level of expression ranged from 0.5 to 6 nkat. The highest activity was displayed by UCS 6 of pGKL2 whereas the lowest level was obtained with UCS2 of pGKL2, all other values were in between. Sequences located 5' upstream the UCSs do not influence expression. As exemplified for UCS5 and UCS10, deletion led to an almost complete loss of expression.

Base Sequence↗
PubMed1996

Yeast killer plasmid pGKL2: molecular analysis of UCS5, a cytoplasmic promoter element essential for ORF5 gene function.

A k2/k1 plasmid gene shuffle system has been used to investigate linear plasmid promoter function in Kluyveromyces lactis. By transplacing various ORF5 deletion constructs from the larger plasmid k2 onto k1, and analysing trans-complementation of an ORF5(0) deletion on k2, a 40 bp k2 fragment, including the UCS motif of ORF5 (UCS5), has been identified as a cis-acting promoter element essential for ORF5 gene function. Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fusion gene using Northern blot analysis and phosphor image technology revealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, UCS5-driven expression of the marker gene was non-repressible by leucine and reduced five- to eight-fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to express low levels of transcript overall, when relative gene copy numbers (one for the nuclear allele versus 50-100 copies for the plasmid-borne LEU2 gene) are taken into account.

Base Sequence↗
PubMed1996

Genetic Variability Among Isolates and Sexual Offspring of the Plant Pathogenic Fungus Calonectria morganii on the Basis of Random Amplification of Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP)

Thirty-two strains of the phytopathogenic mold Cylindrocladium scoparium (perfect state Calonectria morganii) isolated from ericaceous hosts and two specimens from the ATCC were examined by random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Five oligonucleotides were chosen as primers for differentiation of the isolates. RAPD patterns of the ATCC strains differed significantly from those of the field isolates. Diversity among field isolates was low. Results obtained in RFLP analysis, with telomere repeats of Neurospora crassa as a probe, were highly consistent with the RAPD data. Isolates were paired in all possible combinations; fertile perithecia occurred in only one combination, from which ascospores were analyzed by formal genetics and RAPD. A bipolar mechanism of homogenic incompatibility was found. Ascospore-derived strains were much more variable than field isolates. Phylogenetic trees suggested a correlation to the host plants from which the strains were isolated.

Journal Article↗
PubMed1996

Extranuclear expression of the bacterial xylose isomerase (xylA) and the UDP-glucose dehydrogenase (hasB) genes in yeast with Kluyveromyces lactis linear killer plasmids as vectors.

On the basis of the linear killer plasmid pGKL1 from Kluyveromyces lactis, two new linear hybrid plasmids were constructed. One of these, pRSC126, carried the xylA gene from Streptomyces rubiginosus encoding the xylose isomerase. The other linear hybrid molecule, pRSC128, carried the hasB gene of Streptococcus pyogenes encoding the UDP glucose dehydrogenase. Construction was performed in a way that the putative cytoplasmic promoter element of ORF5 of pGKL2 was fused to the coding region of the heterologous genes. After transformation, in vivo recombination led to the establishment of linear hybrid vectors. Though efficiency of expression was low when compared with bacterial systems, cytoplasmic expression of both genes was clearly demonstrated.

Aldose-Ketose Isomerases↗
PubMed1995

An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

Bacillus megaterium↗
PubMed1995

Generation of a stable non-reverting Leu- mutant of Kluyveromyces lactis by gene disruption.

The LEU2 gene coding for beta-isopropylmalate dehydrogenase of the yeast Kluyveromyces lactis strain AWJ137 was disrupted. In the resulting Leu- strain a 0.57 x 10(3)-base pairs PstI/BglII fragment of the LEU2 coding region was replaced by the TRP1 gene of Saccharomyces cerevisiae. The mutant strain was characterized by stability tests and a physical map of the disrupted region was established by restriction-enzyme analysis combined with hybridization experiments. The usefulness of the mutant strain as a recipient was shown by transformation experiments.

3-Isopropylmalate Dehydrogenase↗
PubMed1995

Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement.

An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependant suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.

Bacillus megaterium↗
PubMed1995

Migration of the yeast linear DNA plasmid from the cytoplasm into the nucleus in Saccharomyces cerevisiae.

The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.

Base Sequence↗
PubMed1994

A novel approach to express a heterologous gene on Kluyveromyces lactis linear killer plasmids: expression of the bacterial aph gene from a cytoplasmic promoter fragment without in-phase fusion to the plasmid open reading frame.

Using the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis a new linear hybrid plasmid (pWKL1) was constructed. It consists of pGKL1 into which, in addition to the previously developed cytoplasmically expressible LEU2* selection marker, a phosphotransferase encoding bacterial antibiotic resistance gene has been integrated. In the hybrid plasmid pWKL1 this gene was preceded by a putative transcriptional signal from pGKL2, i.e., the upstream conserved sequence of ORF5 (UCS5). All foreign genes, so far integrated and expressed in linear plasmids, required in addition to a cytoplasmic promoter (UCS) an in phase fusion to the corresponding open reading frame. In this study the UCS5, not containing the translational start of ORF5, and the aph coding region were intentionally not combined in phase, in order to check whether expression can occur under these conditions. Kluyveromyces lactis transformants carrying pWKL1 were highly resistant against the antibiotic geneticine (G418) and exhibited elevated levels of APH activity. Expression of the aph gene was due to the presence of the UCS5 containing fragment (110 bp). Thus, an in phase fusion was not required for expression. This is the first report that, concomitant with a selectable marker gene, foreign DNA can be integrated in a linear plasmid via homologous recombination and more importantly, that in phase fusions are not required generally for expression of heterologous genes in linear plasmids.

Base Sequence↗