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J Altenbuchner

Publications and source records attributed to J Altenbuchner.

At least 55 records · Page 3Linked to original sources
PubMed1994

Analysis of putative DNA amplification genes in the element AUD1 of Streptomyces lividans 66.

The amplifiable AUD1 element of Streptomyces lividans 66 consists of two copies of a 4.7 kb sequence flanked by three copies of a 1 kb sequence. The DNA sequences of the three 1 kb repeats were determined. Two copies (left and middle repeats) were identical: (1009 bp in length) and the right repeat was 1012 bp long and differed at 63 positions. The repeats code for open reading frames (ORFs) with typical Streptomyces codon usage, which would encode proteins of about 36 kD molecular weight. The sequences of these ORFs suggest that they specify DNA-binding proteins and potential palindromic binding sites are found adjacent to the genes. The putative amplification protein encoded by the right repeat was expressed in Escherichia coli.

Base Sequence↗
PubMed1994

Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification.

The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.

Amino Acid Sequence↗
PubMed1994

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends.

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.

Amino Acid Sequence↗
PubMed1994

Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene.

For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens Tü24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tü24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%.

Amino Acid Sequence↗
PubMed1993

A new lambda RES vector with a built-in Tn1721-encoded excision system.

A new lambda replacement vector for construction of genomic libraries was developed which allows the excision of cloned fragments by site-specific recombination from the lambda DNA and conversion into autonomously replicating plasmids. The vector system, derived from lambda EMBL4, is called lambda RES. It contains two recognition sites for site-specific recombination from Tn1721 on both sides of the replacement fragment of lambda EMBL4. Additionally, on one side, there is a plasmid replication origin from Rtsl with a kanamycin-resistance (KmR) marker. DNA fragments in the range of 8-14 kb may be inserted between BamHI or Sall sites in the lambda vector. Efficient excision and conversion of plaque-forming units into KmR colonies are obtained by infection of Escherichia coli strains harbouring Tn1739tnpR on a F' plasmid. Tn1739tnpR is a derivative of Tn1721 with a chloramphenicol-resistance-encoding gene (CmR), the lambda cI repressor gene, and a further copy of the resolvase-encoding tnpR gene under control of the tac promoter.

Bacteriophage lambda↗
PubMed1992

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762.

A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tü24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.

Amino Acid Sequence↗
PubMed1988

Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400.

The scr genes located on plasmid pUR400 and responsible for sucrose (Scr) metabolism of Escherichia coli K12 and other enteric bacteria have been cloned on a 9.3 kb DNA fragment. The different genes were mapped by transposon insertion mutagenesis, by restriction endonuclease and deletion mapping, and the corresponding gene products were identified. Besides the known structural genes scrA, coding for an EnzymeII(Scr) (45 kD) of the phosphoenolypyruvate-dependent phosphotransferase system (PTS), and scrB, coding for a sucrose 6-phosphate hydrolase (invertase) (55 kD), two new structural genes were discovered. Gene scrK apparently codes for an intracellular and ATP-dependent fructokinase (39 kD), while scrY seems to code for a sucrose porin (58 kD) in the outer cell membrane. No genes for an Enzyme III(Scr) of the PTS or for (a) glycosyltransferase(s) were detected. The four genes form an scr operon (gene order, scrK scrY scrA scrB, transcription from K to B), regulated by a repressor (gene scrR, 37 kD) and inducible by sucrose, fructose and fructose-containing oligosaccharides.

DNA Restriction Enzymes↗
PubMed1986

DNA amplification and genetic instability in Streptomyces.

Genetic instability is very common in Streptomyces species, but only affects specific genes in any one strain. It sometimes occurs at high frequency spontaneously, but may be stimulated by treatments such as UV irradiation or intercalating agents. Deletion of genes occurs and may be accompanied by DNA amplifications. It is unlikely that there is plasmid involvement in most cases. Little is yet known about the molecular mechanisms of deletion and DNA amplification. Genetic instability can be a problem during commercial antibiotic production. DNA amplification of cloned genes is potentially useful for achieving both stability and high gene dosage.

Anti-Bacterial Agents↗
PubMed1985

Structure of an amplifiable DNA sequence in Streptomyces lividans 66.

Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.

DNA Restriction Enzymes↗
PubMed1985

Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants.

Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared. These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D). By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities. In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes. Tetracycline acted as an inducer. A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C. Repression of tetRA-linked galactokinase was ca. 80% in the presence of tetRA or tetRC, and ca. 50% in the presence of tetRB. Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants.

Base Sequence↗
PubMed1984

DNA amplification and an unstable arginine gene in Streptomyces lividans 66.

Streptomyces lividans 66 produced spontaneous chloramphenicol-sensitive mutants (CmlS) at a frequency of about 1% of spores. The CmlS mutant strains were very unstable, giving Arg- mutants at frequencies of about 25% of spores. All the Arg- mutants had amplified a particular 5.75 kb DNA fragment into tandem repeats of 250-500 copies per chromosome.

Anti-Bacterial Agents↗
PubMed1983

The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis.

Nucleotide sequences of the homologous tetracycline resistance (tet) determinants of plasmid RP1 and transposon Tn1721 have been determined. Two open reading frames of divergent polarity have been assigned to a regulatory gene (tetR) and a gene encoding a resistance protein (tetA). The intercistronic region contains appropriate regulatory and transcription signals. The tetR gene can code for a protein of 216 amino acids (deduced mol.wt. 23,288) and the tetA gene for a protein of 399 amino acids (deduced mol. wt. 42,205). Based on the deduced amino acid sequence, the tetA proteins of RP1/Tn1721 are 78% homologous with that of pBR322 and 45% homologous with that of Tn10. We conclude that a single tetA gene mediates resistance in each of these tet determinants.

Bacterial Proteins↗
PubMed1983

Transposon Tn1721: site-specific recombination generates deletions and inversions.

Transposon Tn1721 contains genes for transposase (tnpA), resolvase (tnpR) and a resolution site (res). The closely linked loci were localized within a 3.8 kb region their order being res - tnpR - tnpA with res at the translational start of tnpR. Genes tnpR and tnpA have identical transcriptional polarity but independent promoters. The tnpR promoter had 40% of lac promoter efficiency its activity being autoregulated by binding of resolvase to res, as shown by fusion to the galactokinase gene. The weak tnpA promoter was only detectable in the transposase-mediated transposition reaction. Resolvase-catalyzed, site-specific recombination was analyzed in hybrid plasmids with either direct or inverted repeats of res. The rate of the reaction was dependent on the relative orientation of the two sites and on the provision of tnpR in cis or in trans. Direct repeats were rapidly resolved leading to deletions of intervening DNA, if the tnpR gene was provided in cis, but required approximately 60 generations for completion of the reaction, if tnpR was located on a second plasmid (in trans). The reaction involving inverted repeats of res (leading to inversion of intervening DNA) was only detectable if tnpR was furnished in cis. After 50 generations about 10% of plasmid DNA showed the inversion. The reaction was reversible.

Chromosome Deletion↗
PubMed1983

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721.

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.

Base Sequence↗
PubMed1983

Tn1721-encoded tetracycline resistance: mapping of structural and regulatory genes mediating resistance.

The genes encoding inducible tetracycline resistance in Tn1721 were located in a 2.1-kilobase portion of the transposon. Using deletions and insertions, we mapped and characterized two tet genes by their mutant phenotypes. Two tetracycline-inducible polypeptides synthesized in minicells were assigned to the tet genes. The polarity of the tet genes was determined by employing a deletion and a gene fusion which altered the carboxy termini of the polypeptides. The gene responsible for resistance (tetA) encompasses 1,250 base pairs and encodes a membrane-bound protein with an apparent molecular weight of 34,000. The second gene (tetR) encompasses at least 650 base pairs and encodes a soluble 26,000-dalton protein, identified by complementation analysis as the repressor. The two adjacent genes have opposite transcriptional polarity, suggesting that the sites controlling their expression are located in the intercistronic region between tetA and tetR.

Bacterial Proteins↗