Ulf Stahl

The Protein Family of RNA Helicases

RNA helicases represent a large family of proteins that have been detected in almost all biological systems where RNA plays a central role. They are ubiquitously distributed over a wide range of organisms and are involved in nuclear and mitochondrial splicing processes, RNA editing, rRNA processing, translation initiation, nuclear mRNA export, and mRNA degradation. RNA helicases are described as essential factors in cell development and differentiation, and some of them play a role in transcription and replication of viral single-stranded RNA genomes. Comparisons of the conserved sequences reveal a close relationship between them and suggest that these proteins might be derived from a common ancestor. Biochemical studies have revealed a strong dependence of the unwinding activity on ATP hydrolysis. Although RNA helicase activity has only been demonstrated for a few examples yet, it is generally believed that all members of the largest subgroups, the DEAD and DEAH box proteins, exhibit this activity.

Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae

ABSTRACT The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina.

SummaryThe existece of plasmid like DNA was demonstrated in senescent mycelia of Podospora anserina (strain s) by biophysical and electronmicroscopic methods. According to their contour length of about 1.4 and 2.7 μm respectively the molecular weight for the monomer is in the range of 3·106.

Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene.

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate‐decarboxylase (PDC) and increased NAD‐dependent glycerol‐3‐phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20‐fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by‐product formation.

Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, physiological and genetic diversity. Eighteen isolates able to metabolize naphthalene or phenanthrene as sole carbon source were taxonomically affiliated to different subclasses of the Proteobacteria (Sphingomonas spp., Acidovorax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Gram-positive bacteria with low and high DNA G + C content (Paenibacillus sp. and Rhodococcus spp., respectively). Representatives of the genera Pseudomonas and Sphingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon and energy had an active catechol 2,3-dioxygenase (C230). C230 specific activities were very diverse, ranging from 0.1 to 650 mU (mg protein)-1. Pseudomonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradation, by PCR amplification of gene fragments and subsequent hybridization. PCR primers and internal oligonucleotide probes were developed for the specific detection of the genes of Pseudomonas and Sphingomonas strains.

Plasmid-like DNA is part of mitochondrial DNA in Podospora anserina

SummaryAs previously reported, a ccc DNA with a contour length of 0.75 µm and molecular weight of 2.4 kb (termed plasmid-like, p1DNA) is the causative agent of senescence in the fungus Podospora anserina. Its postulated location in mtDNA was proved correct by the following experiments:1.Restriction analysis of mtDNA resulted in different molecular weights for both, juvenile (95 kb) and senescent (30 kb) mtDNA. The construction of a detailed restriction map made evident the fact that senescent mtDNA comprises only a part of its juvenile counterpart.2.Hybridization experiments (Southern blots) between 3H-labelled plDNA and mtDNA cleaved by restriction juvenile mtDNA are homologous to plDNA.3.Fine mapping experiments (construction of restriction maps and heteroduplex experiments) between plDNA integrated into a bacterial vector and its postulated equivalent, derived from juvenile mtDNA and also integrated into a bacterial vector, allowed a precise determination of the site of plDNA insertion within the juvenile mtDNA. All of these data fit into a previously published model in which, during aging, plDNA is excised from mtDNA and becomes autonomous for replication and function.

The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

ABSTRACT We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 μg/ml for Fusarium oxysporum to 200 μg/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.

The onset of senescence is affected by DNA rearrangements of a discontinuous mitochondrial gene in Podospora anserina.

SummaryMapping and transcription studies have revealed that in Podospora anserina the causative agent of senescence, a mitochondrial plasmid (p1DNA), is identical with intronl of the discontinuous gene for cytochrome-c-oxidase subunit 1 (COI), which is 2 kpb from the discontinuous gene for cytochrome b (Cytb). A mitochondrial mutant (ex1) devoid of the COI, but not of the Cytb gene provides longevity. A molecular model for the onset of senescence is presented.