Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae)

A gene (ORFB) from Streptomyces antibioticus (an oleandomycin producer) encoding a large, multifunctional polyketide synthase (PKS) was cloned and sequenced. Its product shows an internal duplication and a close similarity to the third subunit of the PKS involved in erythromycin biosynthesis by Saccharopolyspora erythraea, showing the equivalent nine active site domains in the same order along the polypeptide. An unusual feature of this ORF is the GC content of most of the sequence, which is surprisingly low, for a Streptomyces gene; the large number of codons with T in the third position is particularly striking. The last 800 by of the gene stand out as being normal in their GC content, this region corresponding almost exactly to the thioesterase domain of the gene and suggesting that this domain was a late addition to the PKS. Based on the high degree of similarity between the ORFB product and the third subunit of the erythromycin PKS and the occurrence nearby of a gene conferring oleandomycin resistance, it is possible that this gene might be involved in the biosynthesis of the oleandomycin lactone ring.     

Molecular analysis of the phosphate-specific transport (pst) operon ofPseudomonas aeruginosa

Mithramycin is an antitumor antibiotic synthesized byStreptomyces argillaceus. This producer strain is highly resistant in vivo to mithramycin (MIC 100 µg/ml) but sensitive to the related drugs chromomycin and olivomycin (MIC 10 µg/ml). From a genomic library ofS. argillaceus DNA two cosmid clones were isolated which confer a high level of resistance to mithramycin onS. albus. The resistance genes were mapped by subcloning to a 3.9-kbPstI-PvuII fragment. DNA sequence analysis of this fragment revealed one incomplete and three complete open reading frames. Subcloning experiments demonstrated that resistance to mithramycin is mediated by the genesmtrA andmtrB. ThemtrA gene can potentially encode an ATP-binding protein of the ABC transporter superfamily, containing one nucleotide-binding domain and showing similarity with other ABC transporters involved in resistance to daunorubicin, oleandomycin and tetronasin in their respective producer strains. ThemtrB gene codes for an integral membrane protein with six putative transmembrane helices. A mithramycin-sensitive mutant was generated in a gene replacement experiment by disrupting themtrA gene, thus demonstrating that the system encoded by themtrAB genes is essential for conferring resistance to mithramycin inS. argillaceus.     

An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin

Streptomyces longisporoflavus produces the poly-ketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DN A was cloned in Streptomyces Uvldans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2 M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb Kpnl fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (A TP b inding c assette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.     

Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae)

A gene (ORFB) from Streptomyces antibioticus (an oleandomycin producer) encoding a large, multifunctional polyketide synthase (PKS) was cloned and sequenced. Its product shows an internal duplication and a close similarity to the third subunit of the PKS involved in erythromycin biosynthesis by Saccharopolyspora erythraea, showing the equivalent nine active site domains in the same order along the polypeptide. An unusual feature of this ORF is the GC content of most of the sequence, which is surprisingly low, for a Streptomyces gene; the large number of codons with T in the third position is particularly striking. The last 800 by of the gene stand out as being normal in their GC content, this region corresponding almost exactly to the thioesterase domain of the gene and suggesting that this domain was a late addition to the PKS. Based on the high degree of similarity between the ORFB product and the third subunit of the erythromycin PKS and the occurrence nearby of a gene conferring oleandomycin resistance, it is possible that this gene might be involved in the biosynthesis of the oleandomycin lactone ring.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3′ end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P₄₅₀ monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P₄₅₀-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase. Received: 3 December 1997 / Accepted: 12 May 1998     

Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis

Summary A single gene from Streptomyces tenjimariensis, conferring resistance to kanamycin, apramycin and sisomicin, has been cloned in Streptomyces lividans. The mechanism of resistance involves methylation of 16S RNA in the 30S ribosomal subunit.     

Molecular cloning and characterization of two lincomycin-resistance genes, ImrA and ImrB, from Streptomyces lincolnensls 78–11

Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.     

Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3′ end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P₄₅₀ monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P₄₅₀-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric -ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Construction of Environmental DNA Libraries in Escherichia coli and Screening for the Presence of Genes Conferring Utilization of 4-Hydroxybutyrate

Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive E. coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These E. coli clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced orf1 gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.     

Molecular cloning of the nosiheptide resistance gene from Streptomyces actuosus ATCC 25421

An 8.5 kb BamHI DNA fragment conferring resistance to nosiheptide, a peptide antibiotic of the 'thiostrepton group', was cloned from Streptomyces actuosus ATCC 25421 in Streptomyces lividans 1326. Two BamHI fragments of S. actuosus, the 8.5 kb fragment and an additional 3.0 kb fragment, hybridized with a thiostrepton resistance gene probe (pIJ30). The 8.5 kb fragment showed a relatively low degree of homology with the thiostrepton resistance gene. The restriction map of the nosiheptide resistance gene isolated here was significantly different from the map of the thiostrepton resistance gene previously published.     

Functional characterization of orf6 and orf9 genes involved in the biosynthesis of L-oleandrose from Streptomyces antibioticus Tü99

Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and heterologously expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is in support of proving their functions. Furthermore biosynthetic pathway 5,5-gem-dimethyl-6-deoxyglucose (TDP-Lnoviose) has been proposed.