Friedhelm Meinhardt

Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems

Product removal from aqueous media poses a challenge in biotechnological whole-cell biotransformation processes in which substrates and/or products may have toxic effects. The assignment of an additional liquid solvent phase provides a solution, as it facilitates in situ product recovery from aqueous media. In such two-phase systems, toxic substrates and products are present in the aqueous phase in tolerable but still bioavailable amounts. As a matter of course, adequate organic solvents have to possess hydrophobicity properties akin to substrates and products of interest, which in turn involves intrinsic toxicity of the solvents used. The employment of bacteria being able to adapt to otherwise toxic solvents helps to overcome the problem. Adaptive mechanisms enabling such solvent tolerant bacteria to survive and grow in the presence of toxic solvents generally involve either modification of the membrane and cell surface properties, changes in the overall energy status, or the activation and/or induction of active transport systems for extruding solvents from membranes into the environment. It is anticipated that the biotechnological production of a number of important fine chemicals in amounts sufficient to compete economically with chemical syntheses will soon be possible by making use of solvent-tolerant microorganisms.

The cis–trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism

Isomerization of cis to trans unsaturated fatty acids is a mechanism enabling Gram-negative bacteria belonging to the genera Pseudomonas and Vibrio to adapt to several forms of environmental stress. The extent of the isomerization apparently correlates with the fluidity effects caused, i.e. by an increase in temperature or the accumulation of membrane-toxic organic compounds. Trans fatty acids are generated by direct isomerization of the respective cis configuration of the double bond without a shift of its position. The conversion of cis unsaturated fatty acids to trans is apparently instrumental in the adaptation of membrane fluidity to changing chemical or physical parameters of the cellular environment. Such an adaptive mechanism appears to be an alternative way to regulate membrane fluidity when growth is inhibited, e.g. by high concentrations of toxic substances. The cis-trans isomerase (Cti) activity is constitutively present and is located in the periplasma, it requires neither ATP nor any other cofactor such as NAD(P)H or glutathione, and it operates in the absence of de novo synthesis of lipids. Its independence from ATP is in agreement with the negative free energy of the reaction. cti encodes a polypeptide with an N-terminal hydrophobic signal sequence, which is cleaved off during or shortly after the enzyme is transported across the cytoplasmic membrane to the periplasmic space. A functional heme-binding site of the cytochrome c-type was identified in the predicted Cti polypeptide and very recently, direct evidence was obtained that isomerization does not include a transient saturation of the double bond.

Linear plasmids among eukaryotes: fundamentals and application.

Introduction. Plasmids of eukaryotes have long been a neglected subject. Because they were initially found only in Saccharomyces cerevisiae (Sinclair et al. 1967), they were considered to be genetic elements of limited distribution with a cryptic function. Subsequently plasmids were detected in a plant, Zea mays (Pring et al. 1977), and in a filamentous fungus, Podospora anserina (Stahl et al. 1978). The fungal plasmid turned out to be a circular molecule, as expected in accordance with the generally accepted plasmid configuration (ccc = covalently closed circular). The corn plasmid, however, was the first linear plasmid to be described, though it was to prove antecedent to many others. Circular plasmids of eukaryotes have been reviewed extensively (see Esser ct al. 1986). Linear plasmids, many of which have been detected and analysed more recently, have yet to be reviewed in respect of their common properties and their potential application for research.

Microbial linear plasmids

Abstract 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.

Isolation and Molecular Characterization of Chitinase-Deficient Bacillus licheniformis Strains Capable of Deproteinization of Shrimp Shell Waste To Obtain Highly Viscous Chitin

ABSTRACT Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.

Mechanism of cis-trans Isomerization of Unsaturated Fatty Acids in Pseudomonas putida

We studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of Pseudomonas putida S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c type.

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G1 phase of the cell cycle. Despite its heterotrimeric (αβγ) structure, intracellular expression of its smallest subunit, the γ‐toxin, is alone responsible for the G1 arrest. The α subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR‐mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3Δ), blocked export (chs7Δ) or reduced activation (chs4Δ), combined with mislocalization (chs4Δchs5Δ; chs4Δchs6Δ; chs4Δchs5Δchs6Δ) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII‐polymerized chitin, which amounts to almost 90% of the cells chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells. Copyright

The primary target of the killer toxin from Pichia acaciae is tRNAGln

The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S‐phase of the cell cycle and induces DNA double‐strand breaks (DSBs). Surprisingly, loss of the tRNA‐methyltransferase Trm9 – along with the Elongator complex involved in synthesis of 5‐methoxy‐carbonyl‐methyl (mcm5) modification in certain tRNAs – conferred resistance against PaT. Overexpression of mcm5‐modified tRNAs identified tRNAGln(UUG) as the intracellular target. Consistently, toxin‐challenged cells displayed reduced levels of tRNAGln and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNAGln(UUG). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNAGlu(UUC), affecting its target in a mcm5‐dependent manner, PaT exerts activity also on tRNAGln lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNAGln(UUG), tRNAGln(CUG) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNAGln(CUG) as an additional TRM9‐independent target, overexpression of PaTs tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis

SummaryLinear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5′ ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.

Phylogenetic relationships of linear, protein-primed replicating genomes

SummaryRelative phylogenetic distances were estimated for those linear plasmids for which sequencing data were available by comparing the amino-acid sequences of the putative DNA- and RNA-polymerases, and phylogenetic trees were calculated. The relationships obtained accord well with those indicated by other structural characteristics of these genetic elements. It is obvious that linear plasmids constitute a separate group of genetic traits when compared with those of the adenoviruses. However, an overall relationship to these viruses is evident. Among the linear plasmids at least two main groups can be recognized, namely the cytoplasmically and the mitochondrially localized elements.