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W Wohlleben

Publications and source records attributed to W Wohlleben.

53 records · Page 3Linked to original sources
PubMed1992

A metalloprotease gene from Streptomyces coelicolor 'Müller' and its transcriptional activator, a member of the LysR family.

A metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor 'Müller' DSM3030 were isolated and their DNA sequences determined. The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M(r) of 20,000, which is in good agreement with data derived from DNA sequence analysis. The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family. The regulatory protein (MprR) was shown to bind to the intergenic region between mprR and mprA. It was found to activate transcription of mprA in S. lividans and also in Escherichia coli.

Amino Acid Sequence↗
PubMed1992

Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans.

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.

Bacillus subtilis↗
PubMed1991

Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors.

For the isolation of single stranded plasmid DNA, various E. coli and E. coli-Streptomyces shuttle plasmids were equipped with the phage 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 Streptomyces 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).

Blotting, Southern↗
PubMed1991

Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494.

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.

Anti-Bacterial Agents↗
PubMed1991

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor "Müller".

Streptomyces coelicolor "Müller" DSM3030 excretes a lysozyme comprising both beta-1,4-N-acetyl- and beta-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 lysozyme 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.

Amino Acid Sequence↗
PubMed1990

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine.

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.

Amino Acid Sequence↗
PubMed1989

On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette.

The aminoglycoside-3-O-acetyltransferase-I gene (aacC1) from R plasmids of two incompatibility groups (R1033 [Tn1696], and R135) was cloned and sequenced. In the case of R1033, it was shown that the aacC gene is coded by a precise insertion of 833 bp between the aadA promoter and its structural gene in a Tn21 related transposon (Tn1696). This insertion occurs at the same target sequence as that of the OXA-1 beta-lactamase gene insertion in Tn2603. Upstream of the aacC gene, we found an open reading frame (ORF) which is probably implicated in the site-specific recombinational events involved in the evolution of this family of genetic elements. These results provide additional confirmation of the role of Tn21 elements as naturally occurring interspecific transposition and expression cassettes.

Acetyltransferases↗
PubMed1988

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum.

The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tü494 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 pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene 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 coli as 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 pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

Acetyltransferases↗
PubMed1988

Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli.

Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23. The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tü494. Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment. This fragment was shown to include the Ptt-resistance promoter. Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations. Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase.

Acetyltransferases↗
PubMed1987

Development of a plasmid-cloning system for Streptomyces viridochromogenes Tü494.

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tü494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail. A procedure giving rise to transformation efficiencies of 10(4)-10(5) transformants per microgram DNA was worked out. Several Streptomyces plasmid vectors such as pIJ350, pIJ61, pEB2, pGM4 and pSW2 were tested in S. viridochromogenes. Some of these vectors (pGM4, pEB2) showed a high copy number, whereas the copy number of others (pIJ350, pIJ61 and pSW2) was markedly lower. Under non-selective conditions some of the vectors (pIJ350, pEB2) were not stably maintained, in contrast to the vectors pGM4 and pSW2 which did not require any selection pressure for stable maintenance. Therefore, the plasmid vectors pGM4 and pSW2 derived from endogenous replicons of Streptomyces ghanaensis strains represent appropriate plasmid vehicles for cloning experiments in S. viridochromogenes.

Cloning, Molecular↗
PubMed1987

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction.

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 BglII-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.

Cloning, Molecular↗
PubMed1984

Morphological and molecular characterization of several actinophages isolated from soil which lyse Streptomyces cattleya or S. venezuelae.

Several lytic and lysogenic actinophages were isolated from soil samples infected with Streptomyces cattleya and S. venezuelae. The morphologies and some biological properties of the phages, and the physico-chemical characteristics of their DNAs, were compared. Electron micrographs indicated that all the phage heads were of an icosahedral form, but head size and length of the tail varied. Two of the phages had a broad host range; the other isolates could lyse only a limited number of species. The molecular sizes of the phage DNAs were between 32.2 and 98.5 kb as estimated by electron microscopy and restriction enzyme analysis. The same study also indicated that one of the DNA species contained cohesive ends. The G + C content of the DNAs ranged between 45.1 and 74.2 mol % as estimated from melting studies. Sedimentation velocity experiments implied that several of the phage DNAs were probably heavily glycosylated or methylated. These modifications might explain the partial or slow digestion of some of the DNAs by several of the 23 restriction enzymes tested. Protoplasts of the appropriate Streptomyces strains could be efficiently transfected with phage DNA in the presence of 25% (w/v) polyethylene glycol (mol. wt 6000).

Bacteriophages↗
PubMed1981

RNA polymerase binding sites on the broad host range plasmid RP4.

Binding sites of Escherichia coli RNA polymerase on RP4 plasmid DNA were determined electron microscopically. Comparison of the RNA polymerase binding map and the genetic map of RP4 revealed several strong binding sites outside the well-known RP4 genes. RNA polymerase binding sites for the three antibiotic resistance genes were also detected. Two binding sites were observed for the tra-1 region, whereas the tra-2 and tra-3 regions showed no prominent affinity for RNA polymerase. The genomic regions for the replication origins, oriV (for vegetative replication) and oriT (for transfer replication, equivalent to rlx), both exhibited strong binding to RNA polymerase, as did genomic regions which code for trans-acting replication functions (trfA and trfB).

Binding Sites↗
PubMed1980

Adenine + thymine content of different genes located on the broad host range plasmid RP4.

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.

Adenine↗
PubMed1979

Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent.

The his and nif genes of the P1 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 concomitant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined. The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.

DNA, Bacterial↗
PubMedDate not supplied

Isolation and analysis of moenomycin and its biosynthetic intermediates from Streptomyces ghanaensis (ATCC 14672) wildtype and selected mutants.

Streptomyces ghanaensis (ATCC 14672) produces the phosphoglycolipid antibiotic moenomycin consisting of several components. A solid phase extraction procedure was developed which allowed a rapid isolation of both moenomycin and its biosynthetic intermediates from culture filtrates. Semi-preparative high performance liquid chromatography followed by high performance liquid chromatography-mass spectrometry provided structural data on the different moenomycin components. In order to obtain initial information on the biosynthetic pathway, moenomycin non-producing mutants were isolated. They were shown to release intermediates with shorter lipid chains suggesting that the lipid chain synthesis probably takes place at a later stage of the moenomycin biosynthesis. Based on the biological activity and the analytical data, we assume that a modification and in particular a shorter lipid portion drastically influences the inhibitory activity of this antibiotic.

Anti-Bacterial Agents↗