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Die Verknappung fossiler Ausgangsstoffe sowie Klimaschutz und Importabhängigkeit im Energiesektor sind Herausforderungen, mit denen Europa in den nächsten Jahren konfrontiert sein wird. Europa ist bereits Weltführer bei neuen energieeffizienten Baumaterialien, intelligenten Geräten, alternativen Energiesystemen im Wohnbereich, Isolierung und nachhaltigen Verbraucherprodukten. Verglichen mit anderen Regionen der Welt schneiden die europäischen Gebäude in Bezug auf Energieeffizienz mit am besten ab. Europäische Unternehmen haben außerdem begonnen, neue Technologien in Musterhäuser und Entwicklungen zu integrieren. Diese Stärken und das zur Verfügung stehende Wissen müssen gefördert werden, indem die Marktakzeptanz neuer, energieeffizienter Technologien beschleunigt wird. Eine wachsende Nachfrage wird außerdem zu effizienterer Massenproduktion und somit niedrigeren Preisen für solche Produkte führen.

Das tiefere Verständnis der Zusammenhänge in der Nanodimension ist die Basis für eine moderne Produktentwicklung. Nanomaterialien können interessante, neue – auch neue biologische Eigenschaften haben. Daraus ergibt sich die Frage nach einer möglichen nanospezifischen Toxizität. Sie kann von der spezifischen Oberfläche, der Oberflächenchemie, der Größe oder der Form des Nanomaterials abhängen. Bisher ist eine allgemeine Regel für die Abhängigkeit der Toxizität von den genannten Materialeigenschaften noch nicht bekannt. Spezifische Testverfahren zur Abschätzung der Nanotoxizität existieren noch nicht. Eine Entwicklung von Testmethoden zur Untersuchung der Toxizität von Nanomaterialien bezüglich Umwelt und Gesundheit ist ein Anliegen der BASF. Basierend auf bisherigen Erkenntnissen zur Toxikologie, aber auch unter Berücksichtigung möglicher Expositionen am Arbeitsplatz, ist die Aufnahme sowohl über die Atemwege als auch durch Hautkontakt kritisch zu betrachten. In dem Maß, in dem die Nanotechnologien in konkrete Prozesse und Produkte umgesetzt werden, steigt auch die Beurteilungskompetenz zur Abwägung der Chancen gegenüber möglicher Risiken, die mit der Nutzung neuer Technologien in Form innovativer Produkte und Prozesse verbunden sein können. Die BASF erarbeitet eine wissenschaftlich fundierte Datenbasis zur Beurteilung möglicher Risiken, zu der sowohl die Entwicklung spezifischer Expositionsmessung von Nanomaterialien am Arbeitsplatz und die Gefährdungsbeurteilungen entsprechender Arbeitsplätze, Anwendungs und Produktbewertungen als auch die Untersuchung biophysikalischer Eigenschaften und – damit verbunden – spezieller toxikologischer und ökotoxikologische Prüfungen gehören.

ABSTRACTPhosphinothricin tripeptide (PTT) is a peptide antibiotic produced by Streptomyces viridochromogenesTü494, and it is synthesized by nonribosomal peptide synthetases. The PTT biosynthetic gene cluster contains three peptide synthetase genes: phsA, phsB, and phsC. Each of these peptide synthetases comprises only one module. In neither PhsB nor PhsC is a typical C-terminal thioesterase domain present. In contrast, a single thioesterase GXSXG motif has been identified in the N terminus of the first peptide synthetase, PhsA. In addition, two external thioesterase genes, theAand theB, are located within the PTT biosynthetic gene cluster. To analyze the thioesterase function as well as the assembly of the peptide synthetases within PTT biosynthesis, several mutants were generated and analyzed. A phsAdeletion mutant (MphsA) was complemented with two different phsAconstructs that were carrying mutations in the thioesterase motif. In one construct, the thioesterase motif comprising 45 amino acids of phsAwere deleted. In the second construct, the conserved serine residue of the GXSXG motif was replaced by an alanine. In both cases, the complementation of MphsA did not restore PTT biosynthesis, revealing that the thioesterase motif in the N terminus of PhsA is required for PTT production. In contrast, TheA and TheB might have editing functions, as an interruption of the theAand theBgenes led to reduced PTT production, whereas an overexpression of both genes in the wild type enhanced the PTT yield. The presence of an active single thioesterase motif in the N terminus of PhsA points to a novel mechanism of product release.

ABSTRACTActinoplanes friuliensisproduces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensiswas sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of l-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabAand dabBgenes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.

ABSTRACTActinoplanes friuliensisproduces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcusand Staphylococcusstrains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmAand glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridiumsp. or members of the family Enterobacteriaceae. The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmAand glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanesspp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

The potential of femtosecond time-resolved CARS for the determination of J-dependent line broadening coefficients is demonstrated. With the well-developed femtosecond technique the line broadening coefficients of the H2 Q-branch ro-vibrational transitions are measured in pure H2 and in H2 + He and H2 + N2 mixtures. The results for pure H2 show excellent agreement with the values obtained by other groups using high-resolution CARS in the wavenumber domain. The dependence of the experimental broadening on perturber density also shows good agreement with the behavior of broadening found in the literature: a linear dependence in the case of hydrogen and helium as perturber and explicit non-linear behavior in the case of nitrogen. The role of inhomogeneous broadening in time domain CARS is discussed. Copyright © 2002 John Wiley & Sons, Ltd.

Abstract:: We present a simple and efficient source of slow atoms. From a background vapour loaded magneto-optical trap (MOT), a thin laser beam extracts a continuous jet of cold rubidium atoms. The jet that is typical to leaking MOT systems is created without any optical parts placed inside the vacuum chamber. We also present a simple three dimensional numerical simulation of the atomic motion in the presence of these multiple saturating laser fields combined with the inhomogeneous magnetic field of the MOT. At a pressure of P Rb87 = 10-8 mbar and with a moderate laser power of 10 mW per beam, we generate a flux Φ = 1.3�108 atoms/s with a mean velocity of 14 m/s and a divergence of 10 mrad.

The glutamine synthetase II (GSII, encoded byglnII) activity detectable in crude extracts fromStreptomyces coelicoloris low compared to the activity of glutamine synthetase I (GSI, encoded byglnA) and to that of GSII fromS. viridochromogenes. We have identified and sequenced a 3.9-kbBglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) fromS. coelicolor. BesidesglnII, this region contains four ORFs (orf1–orf4). While homologues oforf1andorf2were also found in theglnIIregion of theS. viridochromogeneschromosome, this was not the case fororf3andorf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that theS. coelicolor glnIIgene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Eσ31. Similar promoter structures were found upstream of theS. viridochromogenes glnIIgene. The involvement ofptringlnIIregulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region ofptr, but not with the promoter region ofglnII. Aptrgene replacement mutant (S. coelicolorIP) was also constructed. RT-PCR analysis of RNA from wild-typeS. coelicolorand the IP mutant demonstrated that expression oforf3depends on Ptr. Thus, the difference in gene organization betweenS. coelicolorandS. viridochromogenesis not responsible for the difference in GSII activity.

ABSTRACTSeven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterraneiDSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyAto -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfAto -Cshowed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

Streptomyces coelicolor “Müller” DSM3030 excretes a lysozyme comprising both ß-1,4-N-acetyl-and ß-1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.

We performed molecular phylogenetic analyses of glutamine synthetase (GS) genes in order to investigate their evolutionary history. The analyses were done on 30 DNA sequences of the GS gene which included both prokaryotes and eukaryotes. Two types of GS genes are known at present: the GSI gene found so far only in prokaryotes and the GSII gene found in both prokaryotes and eukaryotes. Our study has shown that the two types of GS gene were produced by a gene duplication which preceded, perhaps by > 1000 million years, the divergence of eukaryotes and prokaryotes. The results are consistent with the facts that (i) GS is a key enzyme of nitrogen metabolism found in all extant life forms and (ii) the oldest biological fossils date back 3800 million years. Thus, we suggest that GS genes are one of the oldest existing and functioning genes in the history of gene evolution and that GSI genes should also exist in eukaryotes. Furthermore, our study may stimulate investigation on the evolution of "preprokaryotes," by which we mean the organisms that existed during the era between the origin of life and the divergence of prokaryotes and eukaryotes.

The previously isolated non‐phosphinothricin tripeptide producing Streptomyces viridochromogenesgene disruption mutant SP62/2 was used to identify and analyze genes encoding early steps of the phosphinothricin tripeptide biosynthesis. Cross‐feeding and bioconversion experiments between SP62/2 and known non‐phosphinothricin tripeptide producing mutants or presumptive phosphinothricin tripeptide precursors revealed that SP62/2 was blocked in step one or two of the phosphinothricin tripeptide biosynthesis. It was shown that the block in the biosynthesis is due to the integration of a temperature‐sensitive plasmid by illegitimate recombination into the phosphinothricin tripeptide biosynthetic gene cluster. The corresponding region was isolated from the wild‐type. A 2.7‐kb DNA fragment was analyzed comprising three ORFs (ppm, ppd, orfX) which are probably translationally coupled. The ppmgene encodes a protein which is similar to PEP‐phosphomutases and the deduced Ppd product shows similarity to the phosphonopyruvate decarboxylase from Streptomyces wedmorensis.

Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PTT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of glnII encoding a 37.3-kilodalton protein of 343 amino acids. A three- to fourfold increase in gamma-glutamyltransferase activity could be observed in S. lividans transformants carrying the glnII gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by glnII was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, glnA and glnII.

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.

The his and nif genes of the Pl type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA-Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomintant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined.

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenesTü494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65–74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the patgene was found. It encodes a polypeptide of 183 amino acids with an Mrof 20621. The base composition of the patgene is typical for Streptomyces[70.1 mol%(G + C) in total and 93.5 mol%(G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia colias well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus[Thompson et al. EMBO J. 9 (1987) 2519–2523]. However, variations were detected in the 5'-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the patgene was modified and introduced into Nicotiana tabacumby Agrobacterium-mediated leaf-disc transformation. The GTG start codon of patwas replaced by ATG. Subsequently the modified pat-coding region was fused to the 35 S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

The expression of the structural gene (sacB) encoding Bacillus subtilis levansucrase in two gram-positive soil bacteria, Corynebacterium glutamicum ATCC 13032 and Streptomyces lividans 1326, was investigated. sacB expression in the presence of sucrose is lethal to C. glutamicum but not to S. lividans. While S. lividans secretes levansucrase into the medium, we could show that the enzyme is retained by C. glutamicum cells. Our results imply that the sacB gene can be used as a positive selection system in coryneform bacteria.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli- Streptomvces shuttle plasmids were equipped with the f1 replication origin. The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used. In contrast, the transformation of Stmptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form. Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin - tripeptide (PTT).