1. |
Homerova D,
Holländerova Z,
Kormanec J,
Sevcik J,
( 1992 ) Cloning and sequencing of the gene encoding a ribonuclease from Streptomyces aureofaciens CCM3239. PMID : 1398084 : DOI : 10.1016/0378-1119(92)90082-z Abstract >>
A ribonuclease-encoding gene (rnaSa3) from Streptomyces aureofaciens CCM3239 has been isolated and sequenced. The deduced amino acid sequence shows 77% homology with RNase Sa from S. aureofaciens.
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2. |
Kormanec J,
Farkasovský M,
Potúcková L,
( 1992 ) Four genes in Streptomyces aureofaciens containing a domain characteristic of principal sigma factors. PMID : 1452038 : DOI : 10.1016/0378-1119(92)90032-k Abstract >>
Four genes encoding sigma-factor-like proteins, hrdA, hrdB, hrdD, and hrdE, were identified in a Streptomyces aureofaciens genomic library using an oligodeoxyribonucleotide probe encoding a peptide motif homologous to the core-binding domain in sigma factors. The deduced proteins have M(r) values of 43,363, 57,172, 36,591, and 57,565, respectively, and strongly resemble all known principal sigma factors, including possession of the characteristic 'rpoD box'. Transcription analysis of the hrd genes by Northern blot hybridization indicated only the expression of hrdB and hrdD and weak transcription of hrdA. A repetitive region of a pentapeptide tandemly repeated 6 and 4 times was identified in the N-terminal part of HrdB and HrdE, respectively. No such domain was found in any principal sigma factors.
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3. |
Kormanec J,
Farkasovský M,
Potúcková L,
Godár S,
( 1992 ) A gene (hur) from Streptomyces aureofaciens, conferring resistance to hydroxyurea, is related to genes encoding streptomycin phosphotransferase. PMID : 1316866 : DOI : 10.1016/0378-1119(92)90719-6 Abstract >>
A novel gene (hur) conferring resistance to hydroxyurea (HU) in Escherichia coli has been identified in a Streptomyces aureofaciens genomic library. The expression of hur in E. coli was under the control of the external plasmid tet promoter. Sequence analysis of a minimal fragment revealed an open reading frame (ORF) encoding a protein of 340 amino acids with an M(r) of 36,049 and an average hydropathy index of 1.13. The predicted protein product was similar to streptomycin phosphotransferases from Streptomyces glaucescens and Streptomyces griseus (52.4% and 50.8% identity, respectively), but it did not confer resistance to streptomycin or to any of the other aminoglycoside antibiotics tested. It is inferred that hur encodes a phosphotransferase that inactivates HU by phosphorylation of the hydroxy group in the hydroxylamine moiety.
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4. |
Mahishi LH,
Tripathi G,
Rawal SK,
( 2003 ) Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources. PMID : 12608576 : DOI : 10.1078/0944-5013-00161 Abstract >>
Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.
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5. |
Novakova R,
Bistakova J,
Homerova D,
Rezuchova B,
Kormanec J,
( 2002 ) Cloning and characterization of a polyketide synthase gene cluster involved in biosynthesis of a proposed angucycline-like polyketide auricin in Streptomyces aureofaciens CCM 3239. PMID : 12384301 : DOI : 10.1016/s0378-1119(02)00889-2 Abstract >>
A new polyketide gene cluster, aur1, was identified in Streptomyces aureofaciens CCM3239 by using genes for the spore-pigment polyketide synthase of the Streptomyces coelicolor whiE operon as a probe. Sequence analysis of three overlapping DNA fragments (encompassing 15,100 bp) revealed 15 open reading frames, the majority of which showed high similarity to the previously characterized type II polyketide synthase genes. The highest similarity was to three Streptomyces polyketide gene clusters involved in biosynthesis of angucycline antibiotics, jadomycin, urdamycin and landomycin. The proposed S. aureofaciens ketosynthase (Aur1D) was phylogenetically more related to all known ketosynthases for polyketide antibiotics in Streptomyces than to spore-pigment ketosynthases. Interestingly, the aur1 gene cluster contained a gene encoding a proposed malonyl-CoA:ACP transacylase that has not been identified in any of the previously characterized type II polyketide synthase cluster. Transcriptional analysis of aur1 revealed a single promoter upstream the first open reading frame (the aur1A gene) that was active in all stages of differentiation with increased activity at the time of aerial mycelium formation. The aur1 gene cluster was disrupted by a homologous recombination, replacing the three genes (aur1B,C,D) including ketosynthase, with antibiotic resistance marker gene in S. aureofaciens chromosome. Disruption did not affect growth and differentiation; disrupted strain produced spores with wild-type gray-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild-type and aur1-disrupted strains revealed an antibacterial compound that was missing in the mutant. The results indicated a role of the S. aureofaciens aur1 gene cluster in biosynthesis of a polyketide secondary metabolite (which we named auricin), and not in the spore pigment biosynthesis.
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6. |
Sevcik J,
Urbanikova L,
Leland PA,
Raines RT,
( 2002 ) X-ray structure of two crystalline forms of a streptomycete ribonuclease with cytotoxic activity. PMID : 12228255 : DOI : 10.1074/jbc.M208425200 Abstract >>
Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.
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7. |
Laurents D,
Pérez-Cañadillas JM,
Santoro J,
Rico M,
Schell D,
Pace CN,
Bruix M,
( 2001 ) Solution structure and dynamics of ribonuclease Sa. PMID : 11455593 : Abstract >>
We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.
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8. |
Sprusanský O,
Rezuchová B,
Homerová D,
Kormanec J,
( 2001 ) Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase of Streptomyces aureofaciens requires GapR, a member of the AraC/XylS family of transcriptional activators. PMID : 11320132 : DOI : 10.1099/00221287-147-5-1291 Abstract >>
Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is developmentally regulated, and induced by glucose in Streptomyces aureofaciens. A gene, gapR, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators was identified upstream of gap. The gapR gene was constitutively expressed from a single promoter during the course of differentiation. By integrative transformation, via double crossover, a stable null mutant of the gapR gene was obtained. The mutation only slightly affected growth, and had no effect on differentiation of S. aureofaciens. However, transcription of the GAPDH-encoding gap gene was substantially reduced in the S. aureofaciens DeltagapR null mutant, irrespective of carbon source used. Though GAPDH activity was about 1.5-fold lower in the mutant, the substantial enzyme activity remained, suggesting the presence of a second GAPDH which is sufficient to ensure growth. The GapR protein, overproduced in Escherichia coli, was shown to bind upstream of the gap-P promoter region. The results indicate a direct role of GapR in regulation of gap expression in S. aureofaciens.
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9. |
Sprusanský O,
Homérová D,
Sevcíková B,
Kormanec J,
( 1999 ) Cloning of the putative aldehyde dehydrogenase, aldA, gene from Streptomyces aureofaciens. PMID : 10997131 : DOI : 10.1007/bf02816249 Abstract >>
A Streptomyces aureofaciens gene, gap, encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was previously identified. Hybridization studies suggested the presence of a second gap gene in S. aureofaciens. To clone the gene, S. aureofaciens subgenomic library was screened with an oligonucleotide probe encoding a peptide motif conserved in all GAPDH. 3352 bp positive BamHI fragment was identified, the length of which correlated with the hybridization signal. The nucleotide sequence of the fragment was determined, and analysis of the sequence revealed the presence of three open reading frames (ORF). However, none of the genes coded for GAPDH. All three genes formed an operon, consisting of gene orf251, with a high homology to a conserved gene present only in archaeabacteria, and the aldA and adhA genes homologous to various eukaryotic and prokaryotic aldehyde- and alcohol-dehydrogenases, with maximum homology to the phenylacetaldehyde dehydrogenases and arylalcohol dehydrogenases, respectively.
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10. |
Weng M,
Pfeifer O,
Krauss S,
Lingens F,
van Pée KH,
( 1991 ) Purification, characterization and comparison of two non-haem bromoperoxidases from Streptomyces aureofaciens ATCC 10762. PMID : 1783900 : DOI : 10.1099/00221287-137-11-2539 Abstract >>
Two non-haem bromoperoxidases (BPO 1 and BPO 2) were purified from the 7-chlorotetracycline-producing strain Streptomyces aureofaciens ATCC 10762. Both enzymes showed azide-insensitive brominating activity, and bromide-dependent peroxidase activity. BPO 1 was a dimer (Mr 65,000) with subunits of identical size (Mr 31,000). The pI was estimated to be 4.5. The enzyme did not cross-react with antibodies raised against the non-haem bromoperoxidase (Mr 90,000) from S. aureofaciens T?24, a strain that also produces 7-chlorotetracycline. The Mr of BPO 2 was estimated to be 90,000. The enzyme had three identical subunits (Mr 31,000), and its isoelectric point was 3.5, identical with that of the bromoperoxidase from S. aureofaciens T?24. Moreover, BPO 2 was immunologically identical with the bromoperoxidase from S. aureofaciens T?24, although both it and BPO 1 could be distinguished electrophoretically from the latter bromoperoxidase.
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11. |
Bihlmaier C,
Welle E,
Hofmann C,
Welzel K,
Vente A,
Breitling E,
Müller M,
Glaser S,
Bechthold A,
( 2006 ) Biosynthetic gene cluster for the polyenoyltetramic acid alpha-lipomycin. PMID : 16723573 : DOI : 10.1128/AAC.00007-06 PMC : PMC1479109 Abstract >>
The gram-positive bacterium Streptomyces aureofaciens T?117 produces the acyclic polyene antibiotic alpha-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that alpha-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.
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12. |
Sevcik J,
Dodson EJ,
Dodson GG,
( 1991 ) Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 A resolution. PMID : 1654932 : Abstract >>
The crystal structures of ribonuclease from Streptomyces aureofaciens (RNase Sa) and its complex with 3'-guanylic acid (guanosine 3'-monophosphate, 3'-GMP) have been determined by the method of isomorphous replacement. The atomic parameters have been refined by restrained least-squares minimization using data in the resolution range 10.0-1.8 A. All protein atoms and more than 230 water atoms in the two crystal structures have been refined to crystallographic R factors of 0.172 and 0.175 respectively. The estimated r.m.s. error in the atomic positions ranges from 0.2 A for well-defined atoms to about 0.5 A for more poorly defined atoms. There are two enzyme molecules in the asymmetric unit, built independently, and referred to as molecules A and B. The value of the average B factor for protein atoms in both structures is about 19 A2 and for water molecules about 35 A2. Electron density for the substrate analogue 3'-GMP was found only at the active site of molecule A. The density was very clear and the positions of all 3'-GMP atoms were refined with precision comparable to that of the protein.
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13. |
Novakova R,
Homerova D,
Feckova L,
Kormanec J,
( 2005 ) Characterization of a regulatory gene essential for the production of the angucycline-like polyketide antibiotic auricin in Streptomyces aureofaciens CCM 3239. PMID : 16079347 : DOI : 10.1099/mic.0.28019-0 Abstract >>
A gene, aur1P, encoding a protein similar to the response regulators of bacterial two-component signal transduction systems, was identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. Expression of the gene was directed by a single promoter, aur1Pp, which was transcribed at low levels during the exponential phase and induced just before the stationary phase. A divergently transcribed gene, aur1R, has been identified upstream of aur1P, encoding a protein homologous to transcriptional repressors of the TetR family. The aur1P gene was disrupted in the S. aureofaciens CCM 3239 chromosome by homologous recombination. The mutation in the aur1P gene had no effect on growth and differentiation. However, biochromatographic analysis of culture extracts from the S. aureofaciens aur1P-disrupted strain revealed that auricin was not produced in the mutant. This indicated that aur1P is essential for auricin production. Transcription from the previously characterized aur1Ap promoter, directing expression of the first gene, aur1A, in the auricin gene cluster, was dramatically decreased in the S. aureofaciens CCM 3239 aur1P mutant strain. Moreover, the Aur1P protein, overproduced in Escherichia coli, was shown to bind specifically upstream of the aur1Ap promoter region. The results indicated that the Aur1P regulator activates expression of the auricin biosynthesis genes.
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14. |
Ramachander TV,
Rawal SK,
( 2005 ) PHB synthase from Streptomyces aureofaciens NRRL 2209. PMID : 15621415 : DOI : 10.1016/j.femsle.2004.10.034 Abstract >>
An approximately 4.9 kb Sau3A I genomic DNA fragment from the Streptomyces aureofaciens NRRL 2209 aiding in the biosynthesis of PHB in recombinant Escherichia coli has been sequenced and analysed for phaC gene. The putative phaC(Sa) gene of 2 kb is 79.1% GC rich and encodes a 63.5 kDa protein. It expressed under its own promoter and significant PHA synthase activity was detected in the recombinant E. coli. This is the first putative PHA synthase gene reported from a Streptomyces sp. with serine as the active nucleophile in the conserved lipase box. The phaC(Sa) was found in close proximity to a regulatory gene, which apparently regulated the phaC expression.
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15. |
Novakova R,
Bistakova J,
Homerova D,
Rezuchova B,
Feckova L,
Kormanec J,
( 2004 ) Cloning and characterization of a new polyketide synthase gene cluster in Streptomyces aureofaciens CCM 3239. PMID : 15497441 : Abstract >>
We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds.
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16. |
Novakova R,
Bistakova J,
Kormanec J,
( 2004 ) Characterization of the polyketide spore pigment cluster whiESa in Streptomyces aureofaciens CCM3239. PMID : 15365693 : DOI : 10.1007/s00203-004-0720-2 Abstract >>
A spore pigment polyketide gene cluster, whiESa, was cloned from Streptomyces aureofaciens CCM3239 using a probe from the S. coelicolor A3(2) whiE gene cluster. Sequence analysis of a 4,657-bp DNA fragment revealed five open reading frames with the highest similarity to the S. coelicolor A3(2) whiE locus responsible for spore pigment biosynthesis, with conservation of the size and position of the genes. The whiESa gene cluster was disrupted by a homologous recombination in S. aureofaciens CCM3239, replacing the most important whiESaIII gene encoding ketosynthase with a thiostrepton resistance gene. The mutation affected spore pigmentation. In contrast to wild-type grey-pink spore pigmentation, the mutant produced white spores, although overall spore morphology was not affected. Transcriptional analysis of whiESa revealed two divergently oriented promoters, whiESap1 and whiESap2, upstream of the whiESaI and whiESaVIII genes, respectively. Both promoters were developmentally regulated in S. aureofaciens CCM3239. They were induced at the late stages of differentiation, during sporulation of aerial hyphae and were dependent upon early sporulation-specific sigma factor sigma(RpoZ) and putative transcription factor WhiB. The level of the transcript originating from the whiESap2 promoter was substantially reduced in a sigF mutant of S. aureofaciens CCM3239, indicating its dependence upon the late sporulation sigma factor sigma(F). Comparison of the whiE promoters in three different spore pigment polyketide clusters revealed a highly conserved region upstream of the -35 promoter region that may bind a transcriptional regulator.
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17. |
Taechowisan T,
Peberdy JF,
Lumyong S,
( 2004 ) PCR cloning and heterologous expression of chitinase gene of endophytic Streptomyces aureofaciens CMUAc130. PMID : 15486827 : Abstract >>
N/A
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18. |
Sevcik J,
Dauter Z,
Lamzin VS,
Wilson KS,
( 1996 ) Ribonuclease from Streptomyces aureofaciens at atomic resolution. PMID : 15299705 : DOI : 10.1107/S0907444995007669 Abstract >>
Crystals of ribonuclease from Streptomyces aureofaciens diffract to atomic resolution at room temperature. Using synchrotron radiation and an imaging-plate scanner, X-ray data have been recorded to 1.20 A resolution from a crystal of native enzyme and to 1.15 A from a crystal of a complex with guanosine-2'-monophosphate. Refinement with anisotropic atomic temperature factors resulted in increased accuracy of the structure. The R factors for the two structures are 10.6 and 10.9%. The estimated r.m.s. error in the coordinates is 0.05 A, less than half that obtained in the previous analysis at 1.7 A resolution. For the well ordered part of the main chain the error falls to below 0.02 A as estimated from inversion of the least-squares matrix. The two independent molecules in the asymmetric unit allowed detailed analysis of peptide planarity and some torsion angles. The high accuracy of the analysis revealed density for a partially occupied anion in the nucleotide binding site of molecule A in the native structure which was not seen at lower resolution. The anisotropic model allowed correction of the identity of the residue at position 72 from cysteine to threonine. Cys72 SG had been modelled in previous analyses with two conformations. The solvent structure was modelled by means of an automated procedure employing a set of objective criteria. The solvent structure for models refined using different programs with isotropic and anisotropic description of thermal motion is compared.
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19. |
Sevcik J,
Hill CP,
Dauter Z,
Wilson KS,
( 1993 ) Complex of ribonuclease from Streptomyces aureofaciens with 2'-GMP at 1.7 A resolution. PMID : 15299531 : DOI : 10.1107/S0907444992007261 Abstract >>
The crystal structure of a complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with guanosine-2'-monophosphate (2'-GMP) has been refined against synchrotron data recorded from a single crystal using radiation from beamline X31 at EMBL, Hamburg, and an imaging plate scanner. The crystals are in space group P2(1)2(1)2(1) with cell dimensions a = 64.7, b = 78.8 and c = 39.1 A. The structure has two enzyme molecules in the asymmetric unit, complexed with 2'-GMP inhibitor with occupancies of 1 and 2/3 (different to the 3'-GMP complex crystal structure where only one of the two independent RNase Sa molecules binds nucleotide), 492 associated water molecules and one sulfate ion, and was refined using all data between 10.0 and 1.7 A to a final crystallographic R factor of 13.25%. Binding of the base to the enzyme confirms the basis for the guanine specificity but the structural results still do not provide direct evidence of the identity and role of the particular residues involved in the catalytic process. New native RNase Sa data to 1.8 A were recorded to provide a reference set measured under comparable experimental conditions. The crystals are in the same space group and have the same lattice as those of the 2'-GMP complex. The native structure with 423 water molecules was refined in a similar manner to the complex to a final R factor of 13.87%. 1.77 A resolution data were independently measured on a 2'-GMP complex crystal at UCLA using an R-AXIS II image plate scanner mounted on a conventional source. The cell dimensions were essentially the same as above. 2'-GMP was bound more fully to molecule A than to molecule B of the RNase Sa. The structure was refined to an R factor of 14.64% with 388 water molecules. This work follows on from the structure determination of native RNase Sa and its complex with 3'-GMP [Sevcik, Dodson & Dodson (1991). Acta Cryst. B47, 240-253].
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20. |
Pfeifer O,
Pelletier I,
Altenbuchner J,
van Pée KH,
( 1992 ) Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762. PMID : 1527491 : DOI : 10.1099/00221287-138-6-1123 Abstract >>
A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens T?24, 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.
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21. |
Nakano T,
Miyake K,
Endo H,
Dairi T,
Mizukami T,
Katsumata R,
( 2004 ) Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens. PMID : 15215601 : DOI : 10.1271/bbb.68.1345 Abstract >>
For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.
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22. |
Sevcík J,
Dauter Z,
Wilson KS,
( 2004 ) Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2. PMID : 15213380 : DOI : 10.1107/S0907444904009035 Abstract >>
Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. The crystal structures of ribonuclease Sa (RNase Sa) and its complexes with mononucleotides have previously been reported at high resolution. Here, the structures of two crystal forms (I and II) of ribonuclease Sa2 (RNase Sa2) are presented at 1.8 and 1.5 A resolution. The structures were determined by molecular replacement using the coordinates of RNase Sa as a search model and were refined to R factors of 17.5 and 15.0% and R(free) factors of 21.8 and 17.2%, respectively. The asymmetric unit of crystal form I contains three enzyme molecules, two of which have similar structures to those seen for ribonuclease Sa, with Tyr87 at the bottom of their active sites. In the third molecule, Tyr87 has moved substantially: the CA atom moves almost 5 A and the OH of the side chain moves 10 A, inserting itself into the active site of a neighbouring molecule at a similar position to that observed for the nucleotide base in RNase Sa complexes. The asymmetric unit of crystal form II contains two Sa2 molecules, both of which are similar to the usual Sa structures. In one molecule, two main-chain conformations were modelled in the alpha-helix. Finally, a brief comparison is made between the conformations of the Sa2 molecules and those of 34 independent molecules taken from 20 structures of ribonuclease Sa and two independent molecules taken from two structures of ribonuclease Sa3 in various crystal forms.
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23. |
Novakova R,
Rehakova A,
Kutas P,
Feckova L,
Kormanec J,
( 2011 ) The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239. PMID : 21393365 : DOI : 10.1099/mic.0.047795-0 Abstract >>
Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.
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24. |
Novakova R,
Kutas P,
Feckova L,
Kormanec J,
( 2010 ) The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239. PMID : 20466770 : DOI : 10.1099/mic.0.037895-0 Abstract >>
Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.
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25. |
Novakova R,
Odnogova Z,
Kutas P,
Feckova L,
Kormanec J,
( 2010 ) Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in Streptomyces aureofaciens CCM 3239. PMID : 20490753 : DOI : 10.1007/s12223-010-0018-5 Abstract >>
An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.
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26. |
Bauerová-Hlinková V,
Dvorský R,
Perecko D,
Povazanec F,
Sevcík J,
( 2009 ) Structure of RNase Sa2 complexes with mononucleotides--new aspects of catalytic reaction and substrate recognition. PMID : 19558492 : DOI : 10.1111/j.1742-4658.2009.07125.x Abstract >>
Although the mechanism of RNA cleavage by RNases has been studied for many years, there remain aspects that have not yet been fully clarified. We have solved the crystal structures of RNase Sa2 in the apo form and in complexes with mononucleotides. These structures provide more details about the mechanism of RNA cleavage by RNase Sa2. In addition to Glu56 and His86, which are the principal catalytic residues, an important role in the first reaction step of RNA cleavage also seems to be played by Arg67 and Arg71, which are located in the phosphate-binding site and form hydrogen bonds with the oxygens of the phosphate group of the mononucleotides. Their positive charge very likely causes polarization of the bonds between the oxygens and the phosphorus atom, leading to electron deficiency on the phosphorus atom and facilitating nucleophilic attack by O2' of the ribose on the phosphorus atom, leading to cyclophosphate formation. The negatively charged Glu56 is in position to attract the proton from O2' of the ribose. Extended molecular docking of mononucleotides, dinucleotides and trinucleotides into the active site of the enzyme allowed us to better understand the guanosine specificity of RNase Sa2 and to predict possible binding subsites for the downstream base and ribose of the second and third nucleotides.
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27. |
Bekeova C,
Rehakova A,
Feckova L,
Vlckova S,
Novakova R,
Mingyar E,
Kormanec J,
( 2016 ) Characterisation of the genes involved in the biosynthesis and attachment of the aminodeoxysugar D-forosamine in the auricin gene cluster of Streptomyces aureofaciens CCM3239. PMID : 26685675 : DOI : 10.1007/s00253-015-7214-9 Abstract >>
We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The �Gsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the �Gsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.
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28. |
Vértesy L,
Tripier D,
( 1985 ) Isolation and structure elucidation of an alpha-amylase inhibitor, AI-3688, from Streptomyces aureofaciens. PMID : 2581812 : DOI : 10.1016/0014-5793(85)80767-5 Abstract >>
A novel polypeptide inhibitor, AI-3688, which acts upon human pancreatic alpha-amylase, was isolated from fermentation broth of Streptomyces aureofaciens. The purified peptide contains no unusual amino acids. Its Mr is 3936. The primary structure of AI-3688 was elucidated by automatic Edman degradation of the native or modified inhibitor. Two intramolecular cysteines form a disulphide bridge, thus creating a ring structure consisting of 17 amino acids. Strong sequence homology also exists to another microbial alpha-amylase inhibitor, tendamistat (HOE 467). This paper discusses the role of a common partial sequence, -Gln-Ser-Trp-Arg-Tyr-, present in the loop of both inhibitors as the active site of microbial peptide alpha-amylase inhibitors.
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29. |
Kormanec J,
Novakova R,
Mingyar E,
Feckova L,
( 2014 ) Intriguing properties of the angucycline antibiotic auricin and complex regulation of its biosynthesis. PMID : 24265028 : DOI : 10.1007/s00253-013-5373-0 Abstract >>
Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.
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30. |
Zhu T,
Cheng X,
Liu Y,
Deng Z,
You D,
( 2013 ) Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens. PMID : 23800859 : DOI : 10.1016/j.ymben.2013.06.003 Abstract >>
Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the �GctcP strain accumulated about 18.9g/l "clean" TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51��0.01min(-1), while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.
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31. |
( 1997 ) Expression of the Streptomyces aureofaciens glyceraldehyde-3-phosphate dehydrogenase gene (gap) is developmentally regulated and induced by glucose. PMID : 9387234 : DOI : 10.1099/00221287-143-11-3555 Abstract >>
In previous experiments, the Streptomyces aureofaciens gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified. To investigate expression of the gene, S1 nuclease mapping and Northern blot hybridization were performed using RNA prepared from S. aureofaciens cultivated under various conditions. These studies suggested monocistronic organization and developmental regulation of the gene. A single promoter, gap-P, was identified upstream of the gap coding region. In cultures grown on solid medium in the absence of glucose, its transcription was induced at the time of aerial mycelium formation. In addition, gap transcription was also induced in substrate mycelium by glucose. A promoter-bearing DNA fragment was inserted into two promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis.
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32. |
( 1993 ) Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate. PMID : 8396032 : DOI : 10.1111/j.1432-1033.1993.tb18145.x Abstract >>
The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.
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33. |
( 1995 ) A self-defense gene homologous to tetracycline effluxing gene essential for antibiotic production in Streptomyces aureofaciens. PMID : 8534971 : DOI : 10.1271/bbb.59.1835 Abstract >>
By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.
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34. |
( 1994 ) PCR cloning and sequencing of the coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK. PMID : 8125314 : DOI : 10.1016/0378-1119(94)90822-2 Abstract >>
The nucleotide sequence of the PCR-cloned coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK was determined. The deduced 106-amino-acid sequence was compared with three other thioredoxins.
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35. |
( 1993 ) Streptomyces aureofaciens whiB gene encoding putative transcription factor essential for differentiation. PMID : 8506145 : DOI : 10.1093/nar/21.10.2512 PMC : PMC309555 Abstract >>
N/A
|
36. |
( 1994 ) The Streptomyces aureofaciens homologue of the whiG gene encoding a putative sigma factor essential for sporulation. PMID : 8200523 : DOI : 10.1016/0378-1119(94)90612-2 Abstract >>
A putative sigma factor-encoding gene, rpoZ, was cloned from Streptomyces aureofaciens. Its deduced protein product showed high similarity to the putative sigma factor encoded by the Streptomyces coelicolor whiG gene. Disruption of rpoZ blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores.
|
37. |
( 1994 ) Cloning of the putative glycogen branching enzyme gene, glgB, from Streptomyces aureofaciens. PMID : 8068720 : DOI : 10.1016/0304-4165(94)90176-7 Abstract >>
The complete nucleotide sequence of a gene, glgB, encoding a putative glycogen branching enzyme from Streptomyces aureofaciens has been determined. The deduced protein of 764 amino acids with an M(r) of 85,292 shows high similarity to the all bacterial glycogen branching enzymes (43% to 49% amino acid identity), and shares all conserved domains of this group of enzymes.
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38. |
Hecht HJ,
Sobek H,
Haag T,
Pfeifer O,
van Pée KH,
( 1994 ) The metal-ion-free oxidoreductase from Streptomyces aureofaciens has an alpha/beta hydrolase fold. PMID : 7664081 : Abstract >>
The crystal structure of the bromoperoxidase A2 from Streptomyces aureofaciens (ATCC 10762) has been determined by isomorphous replacement and refined to 2.05 A resolution with an R-value of 18.4%. The enzyme catalyzes the bromination of organic compounds in the presence of bromide and peroxide. The structure confirms the absence of cofactors such as metal ions or haem groups and shows the general topology of the alpha/beta hydrolase fold. The active centre is at the end of a deep pocket and includes a catalytic triad of Ser 98, Asp 228 and His 257. The active centre is connected by a narrow tunnel to a second pocket on the enzyme surface.
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39. |
( 1994 ) Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification. PMID : 8012573 : DOI : 10.1099/00221287-140-3-509 Abstract >>
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.
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40. |
Dairi T,
Nakano T,
Aisaka K,
Katsumata R,
Hasegawa M,
( 1995 ) Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline. PMID : 7612997 : DOI : 10.1271/bbb.59.1099 Abstract >>
Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.
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41. |
Potúcková L,
Kelemen GH,
Findlay KC,
Lonetto MA,
Buttner MJ,
Kormanec J,
( 1995 ) A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp. PMID : 7476207 : DOI : 10.1111/j.1365-2958.1995.mmi_17010037.x Abstract >>
A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family. Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis. However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B. subtilis, sigma F affects morphological differentiation in Streptomyces. Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. sigma F is proposed to control the late stages of spore development in Streptomyces.
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42. |
Kormanec J,
Lempelová A,
Farkasovský M,
Homerová D,
( 1995 ) Cloning, sequencing and expression in Escherichia coli of a Streptomyces aureofaciens gene encoding glyceraldehyde-3-phosphate dehydrogenase. PMID : 7489920 : DOI : 10.1016/0378-1119(95)00510-d Abstract >>
The structural gene (gap) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) from Streptomyces aureofaciens (Sa) has been cloned and sequenced. The predicted gap product consists of 332 amino acids (aa) (35,312 Da), and has considerable homology (up to 52% aa identity) with other bacterial and eukaryotic gap genes. Sequence analysis of the regions flanking gap revealed two incomplete open reading frames encoding proteins similar to the AraC family of bacterial transcriptional regulators and delta (5)-3-ketosteroid isomerase. The Sa gap gene was expressed at a high level in Escherichia coli (Ec). Transformation of the Ec strain resulted in an up to eightfold increase in specific GAPDH activity.
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43. |
Shliapnikov SV,
Both V,
Kulikov VA,
Dement'ev AA,
Zelinka J,
( 1987 ) [Extracellular guanyl-specific ribonuclease Sa from the actinomycete Streptomyces aureofaciens. Primary structure and homology with ribonucleases from bacteria and fungi]. PMID : 3118883 : Abstract >>
The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.
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44. |
Twigg FF,
Cai W,
Huang W,
Liu J,
Sato M,
Perez TJ,
Geng J,
Dror MJ,
Montanez I,
Tong TL,
Lee H,
Zhang W,
( 2019 ) Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety. PMID : 30589194 : DOI : 10.1002/cbic.201800762 PMC : PMC6590916 Abstract >>
Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.
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45. |
Shlyapnikov SV,
Both V,
Kulikov VA,
Dementiev AA,
Sevcík J,
Zelinka J,
( 1986 ) Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens. PMID : 3098582 : DOI : 10.1016/0014-5793(86)81138-3 Abstract >>
Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.
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46. |
( 2013 ) The gene cluster aur1 for the angucycline antibiotic auricin is located on a large linear plasmid pSA3239 in Streptomyces aureofaciens CCM 3239. PMID : 23373695 : DOI : 10.1111/1574-6968.12095 Abstract >>
We previously identified a polyketide synthase gene cluster, aur1, responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. A sequence analysis of the aur1 flanking regions revealed the presence of several genes encoding proteins homologous to those for Streptomyces linear plasmid replication, partitioning and telomere-binding. Pulse-field gel electrophoresis detected the single, 240-kb linear plasmid, pSA3239, in S. aureofaciens CCM3239. The presence of the auricin cluster in pSA3239 was confirmed by several approaches. In addition to aur1, pSA3239 also carries a large number of regulatory genes, and two gene clusters involved in the production of secondary metabolites: the aur2 cluster for an unknown secondary metabolite and the bpsA cluster for the blue pigment indigoidine.
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47. |
( 2013 ) Recombinant production and characterization of an N-Acyl-D-amino acid amidohydrolase from Streptomyces sp. 64E6. PMID : 23264153 : DOI : 10.1007/s11274-012-1245-5 Abstract >>
N-Acyl-D-amino acid amidohydrolases (D-aminoacylases) are often used as tools for the optical resolution of D-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding D-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)D-Phe (0.05-6.32 �gmol min(-1) mg(-1)) under conditions without CoCl2. Addition of 1 mM CoCl2 enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the D-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 �gmol min(-1) mg(-1) for N-Ac-D-Phe and N-Ac-D-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. D-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0-9.0. It was stable at pH 5.5-9.0 up to 30 �XC. The enzyme hydrolyzed various N-acetyl-D-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-D-Phe was 2.1-fold higher than that toward N-Ac-D-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.
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48. |
( 1999 ) The Streptomyces aureofaciens homologue of the sporulation gene whiH is dependent on rpoZ-encoded sigma factor. PMID : 9931449 : DOI : 10.1016/s0167-4781(98)00258-9 Abstract >>
Using the method for the identification of promoters recognized by a sporulation specific sigma factor RpoZ, we identified a promoter in Streptomyces aureofaciens, directing expression of a gene having high sequence similarity (83% amino acid identity) to sporulation transcription factor WhiH of Streptomyces coelicolor. High-resolution S1-nuclease mapping using RNA prepared from S. aureofaciens from various developmental stages showed high similarity of PwhiH promoter to the consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in S. aureofaciens strain with rpoZ-disrupted gene. The results suggest that the PwhiH promoter is recognized by sigma factor RpoZ in S. aureofaciens.
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49. |
( 1998 ) Structural investigation of the cofactor-free chloroperoxidases. PMID : 9642069 : DOI : 10.1006/jmbi.1998.1802 Abstract >>
The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.
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50. |
( 1998 ) Recognition of RNase Sa by the inhibitor barstar: structure of the complex at 1.7 A resolution. PMID : 9757110 : DOI : 10.1107/s0907444998004429 Abstract >>
We report the 1.7 A resolution structure of RNase Sa complexed with the polypeptide inhibitor barstar. The crystals are in the hexagonal space group P65 with unit-cell dimensions a = b = 56.9, c = 135.8 A and the asymmetric unit contains one molecule of the complex. RNase Sa is an extracellular microbial ribonuclease produced by Streptomyces aureofaciens. Barstar is the natural inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens. It inhibits RNase Sa and barnase in a similar manner by steric blocking of the active site. The structure of RNase Sa is very similar to that observed in crystals of the native enzyme and its complexes with nucleotides. Barstar retains the structure found in its complex with barnase. The accessible surface area of protein buried in the complex is about 300 A2 smaller and there are fewer hydrogen bonds in the enzyme-inhibitor interface in RNase Sa-barstar than in barnase-barstar, providing an explanation of the reduced binding affinity in the former. Previous studies of barstar complexes have used mutants of the inhibitor and this is the first structure which includes wild-type barstar.
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51. |
( 1998 ) Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. PMID : 9819211 : DOI : 10.1021/bi9815243 Abstract >>
The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.
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( 1998 ) Isolation and purification of two novel streptomycete RNase inhibitors, SaI14 and SaI20, and cloning, sequencing, and expression in Escherichia coli of the gene coding for SaI14. PMID : 9515932 : PMC : PMC107063 Abstract >>
Two new RNase inhibitors, SaI14 (Mr, approximately 14,000) and SaI20 (Mr, approximately 20,000), were isolated and purified from a Streptomyces aureofaciens strain. The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.
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( 1998 ) The gene downstream of Streptomyces aureofaciens whiB encodes a large protein with proposed transmembrane localization, and is induced by glucose. PMID : 9565673 : DOI : 10.1016/s0167-4781(98)00013-x Abstract >>
The sequence analysis of the region downstream of the Streptomyces aureofaciens whiB sporulation gene revealed a long open reading frame (1219 amino acids; Mr 128 209) encoding protein with potential transmembrane structure. By integrative transformation, via double cross-over, a stable null mutant of the gene, orf1219, was prepared. This mutation appeared to have no obvious effect on vegetative growth and differentiation. In vitro and in vivo transcriptional analysis of the downstream gene revealed a single apparent promoter induced by glucose.
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( 1998 ) A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ. PMID : 9479043 : DOI : 10.1016/s0378-1119(97)00645-8 Abstract >>
We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).
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