| 1. |
Dammann T,
Wohlleben W,
( 1992 ) A metalloprotease gene from Streptomyces coelicolor 'M?ller' and its transcriptional activator, a member of the LysR family. PMID : 1406267 : DOI : 10.1111/j.1365-2958.1992.tb01402.x Abstract >>
A metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor 'M?ller' DSM3030 were isolated and their DNA sequences determined. The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M(r) of 20,000, which is in good agreement with data derived from DNA sequence analysis. The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family. The regulatory protein (MprR) was shown to bind to the intergenic region between mprR and mprA. It was found to activate transcription of mprA in S. lividans and also in Escherichia coli.
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2. |
Takano E,
Gramajo HC,
Strauch E,
Andres N,
White J,
Bibb MJ,
( 1992 ) Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). PMID : 1435258 : DOI : 10.1111/j.1365-2958.1992.tb01459.x Abstract >>
Transcription of redD, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redD expression was followed by transcription of redX, a biosynthetic structural gene, and the appearance of the antibiotic in the mycelium, and coincided with the intracellular appearance of ppGpp. However, ppGpp production elicited either by nutritional shift-down of, or addition of serine hydroxamate to, exponentially growing cultures had no stimulatory effect on redD transcription. The presence of redD on a multicopy plasmid resulted in elevated levels of the redD transcript and production of redX and undecylprodigiosin during exponential growth; the normal growth-phase-dependent production of undecylprodigiosin appeared to be mediated entirely through the redD promoter, which shows limited similarity to the consensus sequence for the major class of eubacterial promoters.
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3. |
Davis NK,
Chater KF,
( 1992 ) The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation. PMID : 1316997 : DOI : 10.1007/bf00266237 Abstract >>
A non-sporulating mutant (whiB218) of Streptomyces coelicolor A3(2), which proved to contain a deletion of more than 5 kb of DNA including whiB, was complemented by a small cloned DNA fragment, deduced from DNA sequencing to encode a protein of only 87 amino acids. This protein would bear some similarities to transcription factors, including an acidic, somewhat amphipathic alpha-helical region predicted near its N-terminus, and a basic alpha-helical region predicted at its C-terminus. A point mutation (whiB70) giving a phenotype indistinguishable from that of the whiB218 deletion mutant would cause a leucine to proline change at the start of the latter region. The whiB homologue from the closely related species S. lividans differed at only one base from its S. coelicolor counterpart, and would specify an identical polypeptide.
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4. |
Martínez-Costa OH,
Zalacaín M,
Holmes DJ,
Malpartida F,
( 2003 ) The promoter of a cold-shock-like gene has pleiotropic effects on Streptomyces antibiotic biosynthesis. PMID : 12670683 : DOI : 10.1016/S0378-1097(03)00101-0 Abstract >>
We have isolated a Streptomyces hygroscopicus chromosomal DNA fragment able to induce production of the blue-pigmented antibiotic actinorhodin in Streptomyces lividans. The 1.9-kb fragment contains four orfs (orf1-4) of which only orf2 and orf3 were complete. The minimal region involved in activation of actinorhodin production is limited to 165 bp corresponding to the promoter region of orf3. The truncated Orf1 show homologies with threonine synthases, Orf2 is similar to other proteins of unknown function, Orf3 (here named Csp1) is homologous to cold-shock-induced proteins of the Csp family, and Orf4 encodes the N-terminal region of GroEL2. Transcription of csp1 seems to be subjected to temporal control but is not obviously induced by cold shock. Interestingly, the csp1-groEL2 region pleiotropically regulates the production of antibiotics from Streptomyces coelicolor and Streptomyces nodosus.
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5. |
Bao K,
Cohen SN,
( 2003 ) Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication. PMID : 12651895 : DOI : 10.1101/gad.1060303 PMC : PMC196017 Abstract >>
Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.
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6. |
Keatinge-Clay AT,
Shelat AA,
Savage DF,
Tsai SC,
Miercke LJ,
O'Connell JD,
Khosla C,
Stroud RM,
( 2003 ) Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase. PMID : 12575934 : Abstract >>
Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.
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7. |
Sciara G,
Kendrew SG,
Miele AE,
Marsh NG,
Federici L,
Malatesta F,
Schimperna G,
Savino C,
Vallone B,
( 2003 ) The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis. PMID : 12514126 : DOI : 10.1093/emboj/cdg031 PMC : PMC140106 Abstract >>
ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.
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8. |
Wilkinson CJ,
Hughes-Thomas ZA,
Martin CJ,
Böhm I,
Mironenko T,
Deacon M,
Wheatcroft M,
Wirtz G,
Staunton J,
Leadlay PF,
( 2002 ) Increasing the efficiency of heterologous promoters in actinomycetes. PMID : 12125822 : Abstract >>
An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.
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9. |
Viollier PH,
Nguyen KT,
Minas W,
Folcher M,
Dale GE,
Thompson CJ,
( 2001 ) Roles of aconitase in growth, metabolism, and morphological differentiation of Streptomyces coelicolor. PMID : 11325949 : DOI : 10.1128/JB.183.10.3193-3203.2001 PMC : PMC95221 Abstract >>
The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184-3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in Streptomyces coelicolor development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned acoA gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an acoA disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an acoA citA mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription of acoA was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.
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10. |
Viollier PH,
Minas W,
Dale GE,
Folcher M,
Thompson CJ,
( 2001 ) Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis. PMID : 11325948 : DOI : 10.1128/JB.183.10.3184-3192.2001 PMC : PMC95220 Abstract >>
Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.
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11. |
Bao K,
Cohen SN,
( 2001 ) Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces. PMID : 11410532 : DOI : 10.1101/gad.896201 PMC : PMC312717 Abstract >>
Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5' DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.
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12. |
Molle V,
Bibb MJ,
( 2000 ) sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2). PMID : 10913095 : DOI : 10.1128/jb.182.16.4606-4616.2000 PMC : PMC94633 Abstract >>
Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced whi strains (N. J. Ryding et al., J. Bacteriol. 181:5419-5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the "bald" phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for sigma(BldN) holoenzyme in vitro.
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13. |
Molle V,
( 2000 ) Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages. PMID : 10931278 : DOI : 10.1046/j.1365-2958.2000.01977.x Abstract >>
whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although bldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for bldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. bldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.
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14. |
Folcher M,
Morris RP,
Dale G,
Salah-Bey-Hocini K,
Viollier PH,
Thompson CJ,
( 2001 ) A transcriptional regulator of a pristinamycin resistance gene in Streptomyces coelicolor. PMID : 11050092 : DOI : 10.1074/jbc.M007690200 Abstract >>
Pip is a pristinamycin-induced transcriptional regulator protein detected in many Streptomyces species by its ability to specifically bind sequence motifs within the promoter of a Streptomyces pristinaespiralis multidrug resistance gene (ptr). To investigate the possible role of Pip in regulating multidrug resistance, it was purified from a genetically characterized species, Streptomyces coelicolor, utilizing an affinity matrix of the ptr promoter conjugated to magnetic beads. Reverse genetics identified the corresponding locus and confirmed that it encoded Pip, a protein belonging to the TetR family of procaryotic transcriptional repressors. Pip binding motifs were located upstream of the adjacent gene pep, encoding a major facilitator antiporter homologous to ptr. In vivo analysis of antibiotic susceptibility profiles demonstrated that pep conferred elevated levels of resistance only to pristinamycin I (PI), a streptogramin B antibiotic having clinical importance. Purified recombinant Pip was a dimer (in the presence or absence of PI) and displayed a high affinity for its palindromic binding motifs within the ptr promoter and the upstream region of pep. The Pip/ptr promoter complex was dissociated by PI but not by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers. These procaryotic regulatory elements served as the basis for the development of systems allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including pristinamycin, virginiamycin, and Synercid(R)).
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15. |
Bruton CJ,
Schneider D,
( 2000 ) Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2). PMID : 10821190 : DOI : 10.1007/s004380051200 Abstract >>
DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pinmelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the alpha-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose).
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16. |
Gomez J,
Bishai WR,
Soliveri JA,
( 2000 ) Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes. PMID : 10708372 : DOI : 10.1099/00221287-146-2-333 Abstract >>
The whiB sporulation gene of Streptomyces coelicolor was shown [Davis, N. K. & Chater, K. F. (1992). Mol Gen Genet 232, 351-358] to encode a small, cysteine-rich putative transcription factor unlike any that had been described previously. The large database of DNA sequences of mycobacteria (like Streptomyces, members of the Actinomycetales) has revealed a family of genes encoding proteins related to WhiB. Mycobacterium tuberculosis contains at least six such genes (whiB homologues in mycobacteria: whmA-F) and a likely seventh, whmG. Using conserved features of Whm proteins, a PCR-based approach led to the discovery that S. coelicolor A3(2) contains several similar genes. Cloning and sequencing of these whiB-like (wbI) genes revealed likely orthologues of four of the whm genes of M. tuberculosis. In all, S. coelicolor contains at least five wbI genes in addition to whiB itself. All five were shown by RT-PCR to be transcribed. A Southern blotting survey using each wbI gene as a probe showed that nearly all of a series of representatives of ten actinomycete genera (including morphologically simple organisms) contain close homologues of several wbI genes, suggesting that the ancient progenitor of all these organisms already contained a family of such genes, which have not been found in any other organisms.
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17. |
Lee EJ,
Cho YH,
( 2000 ) A developmentally regulated catalase required for proper differentiation and osmoprotection of Streptomyces coelicolor. PMID : 10632885 : DOI : 10.1046/j.1365-2958.2000.01685.x Abstract >>
Streptomyces coelicolor produces at least three catalases, the expression of which varies under different conditions. We characterized a gene (catB) for developmentally controlled catalase of 779 amino acids (83408 Da), homologous to KatE of Escherichia coli and Bacillus subtilis. Expression of the catB gene increased at the stationary phase in liquid culture and after the onset of differentiation on solid culture. It was also increased by osmotic treatments. Transcription was initiated from a promoter (catBp), whose sequence (ATGCCTCG-N13-GGGTAC) resembled promoters recognized by sigmaB of B. subtilis. CatB protein underwent proteolytic cleavage of its N-terminal 95 amino acids and was secreted to the medium when cells sporulated. Disruption of the catB gene caused impairment in the formation of aerial mycelium and reduction in the synthesis of undecylprodigiosin. On the contrary, hyperproduction of actinorhodin was observed in accordance with the increase in actII-ORF4 transcription. In addition, catB mutant became hypersensitive to osmotic stresses. These results suggest that regulated synthesis of CatB protein is necessary to ensure proper differentiation as well as to protect S. coelicolor cells against osmotic stresses.
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18. |
Kawase T,
Tanabe T,
Mitsutomi M,
Kanai R,
Watanabe T,
( 1999 ) Family 19 chitinases of Streptomyces species: characterization and distribution. PMID : 10627034 : DOI : 10.1099/00221287-145-12-3353 Abstract >>
Chitinase C from Streptomyces griseus HUT6037, described in 1997, is the first family 19 chitinase found in an organism other than higher plants. In this study, some properties of chitinase C were compared with those of family 18 bacterial chitinases, and the distribution of family 19 chitinases in Streptomyces species was investigated. The specific hydrolysing activity of chitinase C against soluble and insoluble chitinous substrates was markedly higher than those of bacterial family 18 chitinases. Chitinase C exhibited marked antifungal activity, whereas the other bacterial chitinases examined had no antifungal activity. Chitinase C was insensitive to allosamidin, whereas the family 18 bacterial chitinases were sensitive. Taking advantage of this insensitivity to allosamidin, a search was made for family 19 chitinases in various Streptomyces species. Chitinases insensitive to allosamidin were detected in the culture supernatants of all tested Streptomyces species. Southern hybridization analysis using a labelled DNA fragment corresponding to the catalytic domain of chitinase C strongly suggested that these species have genes similar to the chiC gene of S. griseus HUT6037. DNA fragments corresponding to the major part of the catalytic domains were amplified by PCR. The amplified fragments encoded amino acid sequences very similar to that of the corresponding region of chitinase C. Therefore, it was concluded that Streptomyces species generally possess family 19 chitinases which are very similar to chitinase C. Comparison of their amino acid sequences with those of plant family 19 chitinases revealed that Streptomyces family 19 chitinases are class IV type in terms of the presence and positions of deletions of amino acid sequences which are characteristic of plant class IV chitinases.
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19. |
Aoyagi N,
Ogawara H,
( 1999 ) Sequences and evolutionary analyses of eukaryotic-type protein kinases from Streptomyces coelicolor A3(2). PMID : 10627033 : DOI : 10.1099/00221287-145-12-3343 Abstract >>
Four eukaryotic-type protein serine/threonine kinases from Streptomyces coelicolor A3(2) were cloned and sequenced. To explore evolutionary relationships between these and other protein kinases, the distribution of protein serine/threonine kinase genes in prokaryotes was examined with the TFASTA program. Genes of this type were detected in only a few species of prokaryotes and their distribution was uneven; Streptomyces, Mycobacterium, Synechocystis and Myxococcus each contained more than three such genes. Homology analyses by GAP and Rdf2 programs suggested that some kinases from one species were closely related, whilst others were only remotely related. This was confirmed by examining phylogenetic trees constructed by the neighbour-joining and other methods. For each species, analysis of the coding regions indicated that the G+C content of protein kinase genes was similar to that of other genes. Considered with the fact that in phylogenetic trees the amino acid sequences of STPK from Aquifex aeolicus and some other eukaryotic-type protein kinases in prokaryotes form a cluster with protein kinases from eukaryotes, this suggests that the eukaryotic-type protein kinases were present originally in both prokaryotes and eukaryotes, but that most of these genes have been lost during the evolutionary process in prokaryotes because they are not needed. This conclusion is supported by the observation that the prokaryotes retaining several of these kinases undergo complicated morphological and/or biochemical differentiation.
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20. |
Rodríguez E,
( 1999 ) Genetic and biochemical characterization of the alpha and beta components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). PMID : 10589718 : DOI : 10.1099/00221287-145-11-3109 Abstract >>
Two genes, accA1 and accA2, with nearly identical nucleotide sequences were cloned from Streptomyces coelicolor A3(2). The deduced amino acid sequences of the product of these two genes showed high similarity to BcpA2 of Saccharopolyspora erythraea and other biotin-containing proteins from different organisms assumed to be the alpha subunit of a propionyl-CoA carboxylase. A gene, pccB, encoding the carboxyl transferase subunit of this enzyme complex was also characterized. Strains disrupted in accA1 did not show any change in acetyl- or propionyl-CoA carboxylase activity, whilst cell-free extracts of a pccB mutant strain contained a reduced level of propionyl-CoA carboxylase. No mutants in accA2 could be isolated, suggesting that the gene may be essential. Heterologous expression of accA1, accA2 and pccB in Escherichia col and in vitro reconstitution of enzyme activity confirmed that PccB is the beta subunit of a propionyl-CoA carboxylase and that either AccA1 or AccA2 could act as the alpha component of this enzyme complex. The fact that accA2 mutants appear to be inviable suggests that this gene encodes a biotinylated protein that might be shared with other carboxyl transferases essential for the growth of S. coelicolor.
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21. |
Potter CA,
Craster HL,
( 1999 ) End-product control of expression of branched-chain amino acid biosynthesis genes in Streptomyces coelicolor A3(2): paradoxical relationships between DNA sequence and regulatory phenotype. PMID : 10517590 : DOI : 10.1099/00221287-145-9-2375 Abstract >>
The branched-chain protein amino acids isoleucine, valine and leucine can provide precursors for synthesis of complex polyketide secondary metabolites in streptomycetes; therefore the regulation of their own synthesis is of interest. DNA sequences upstream of ilvBNC, ilvD, leuA, leuB, ilvE and leuCD in Streptomyces coelicolor A3(2) have been obtained in this laboratory or as part of the S. coelicolor genome sequencing project. Upstream of ilvB and leuA, typical features of classical attenuator systems can be discerned, in particular hypothetical short ORFs with runs of Ile/Val/Leu and Leu codons, respectively. No such features are apparent upstream of other genes or gene clusters present. All five upstream regions were fused to xylE (encoding catechol dioxygenase, CO) as a reporter gene in the SCP2*-based low-copy-number vector pIJ2839. All wild-type regions showed strong depression of CO activity in the presence of all three branched-chain amino acids whether or not the attenuation features were present. By site-directed mutagenesis, the Ile/Val/Leu and Leu triplets in the putative attenuator peptides for ilvB and leuA were replaced by ones for other amino acids. In the case of ilvB, this had no effect at all; for leuA, the wild-type regulatory phenotype persisted in at least some experiments. It was concluded that (i) an unknown regulatory mechanism must be operating in the ilv/leu system of S. coelicolor A3(2) in place of classical attenuation; and (ii) it is unsafe to infer the functioning of a regulatory mechanism from sequence homologies alone.
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22. |
González-Cerón G,
Kieser HM,
Valdez F,
( 1999 ) The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator. PMID : 10517589 : DOI : 10.1099/00221287-145-9-2365 Abstract >>
A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved -10 and -35 regions, as well as additional conserved sequences upstream of the -35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.
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23. |
Davies RA,
Lobo S,
Morgenstern MR,
McArthur HA,
Fedechko RW,
Skinner DD,
Denoya CD,
Zhang YX,
( 1999 ) Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production. PMID : 10517585 : DOI : 10.1099/00221287-145-9-2323 Abstract >>
The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoA-derived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.
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24. |
Falke D,
Fink D,
( 1999 ) Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase. PMID : 10517584 : DOI : 10.1099/00221287-145-9-2313 Abstract >>
An internal adenylyltransferase gene (glnE) fragment from Streptomyces coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of glnE. glnE from S. coelicolor lies on the Asel-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of glnE in S. coelicolor, glnE (S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the gamma-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.
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25. |
Leibovitz E,
( 1999 ) A putative two-component signal transduction system regulates sigmaE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2). PMID : 10411727 : DOI : 10.1046/j.1365-2958.1999.01452.x Abstract >>
The extracytoplasmic function (ECF) sigma factor, sigmaE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of sigmaE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE; orf202, cseB and cseC (cse, control of sigE). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+-deficient cultures than in high Mg2+-grown cultures. We propose a model in which the CseB/CseC two-component system modulates activity of the sigE promoter in response to signals from the cell envelope.
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26. |
Ratnatilleke A,
Grubelnik-Leiser A,
Zerbe-Burkhardt K,
Vrijbloed JW,
( 1999 ) Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis: influence on polyketide antibiotic biosynthesis. PMID : 10482499 : PMC : PMC94078 Abstract >>
The coenzyme B(12)-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.
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27. |
Hood DW,
Heidstra R,
Hodgson DA,
( 1999 ) The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression. PMID : 10361288 : DOI : 10.1046/j.1365-2958.1999.01407.x Abstract >>
Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.
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28. |
Chung HJ,
Roe JH,
Choi JH,
Suh B,
( 1999 ) Duplicate genes for Fe-containing superoxide dismutase in Streptomyces coelicolor A3(2). PMID : 10231572 : DOI : 10.1016/s0378-1119(99)00088-8 Abstract >>
Streptomyces coelicolor M?ller contains two types of superoxide dismutase (SOD) containing Ni (encoded by sodN) or Fe (encoded by sodF). Unlike a single species of Fe-containing SOD in M?ller strain, multiple forms of FeSODs were detected in S. coelicolor A3(2) strain by activity staining and Western blot analysis. Genomic Southern hybridization suggested the presence of at least two copies of the sodF-like gene in A3(2). Two different genes for FeSOD (sodF1 and sodF2) were isolated from the phage library of A3(2) genome. The nucleotide sequence of the sodF1 coding region was identical with the unique sodF gene from M?ller while that of sodF2 shared 88% identity. The gene products of sodF1 and sodF2 were identified by activity staining and immunoblot analysis. Expression from the sodF1 gene was repressed by nickel as sensitively as M?ller sodF, suggesting the presence of Ni-responsive regulatory site within the region shared by the two genes. Among 12 other Streptomyces species examined, only S. fradiae contained two FeSOD-like polypeptides. We postulate that the additional copy of the sodF gene (sodF2) was provided by the horizontal transfer from remotely related bacteria.
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29. |
Mazodier P,
Servant-Moisson P,
Viala J,
Grandvalet C,
( 1999 ) Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces. PMID : 10320574 : DOI : 10.1046/j.1365-2958.1999.01364.x Abstract >>
The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.
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30. |
Kormanec J,
Sevcíková B,
Barák I,
( 1999 ) A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2). PMID : 10188243 : DOI : 10.1111/j.1574-6968.1999.tb13463.x Abstract >>
An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.
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31. |
Caballero JL,
Martinez E,
Malpartida F,
Hopwood DA,
( 1991 ) Organisation and functions of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. PMID : 1766437 : DOI : 10.1007/bf00280297 Abstract >>
Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and -ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.
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32. |
van Wezel GP,
Vijgenboom E,
Bosch L,
( 1991 ) A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA. PMID : 1715981 : DOI : 10.1093/nar/19.16.4399 PMC : PMC328626 Abstract >>
S. coelicolor A3(2) contains six ribosomal RNA operons. Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons. Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon-containing bands. The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical. Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD. Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region.
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33. |
Kim IK,
Lee CJ,
Kim MK,
Kim JM,
Kim JH,
Yim HS,
Cha SS,
Kang SO,
( 2006 ) Crystal structure of the DNA-binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2). PMID : 16689794 : DOI : 10.1111/j.1365-2958.2006.05176.x Abstract >>
BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.
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34. |
Mazurakova V,
Sevcikova B,
Rezuchova B,
Kormanec J,
( 2006 ) Cascade of sigma factors in streptomycetes: identification of a new extracytoplasmic function sigma factor sigmaJ that is under the control of the stress-response sigma factor sigmaH in Streptomyces coelicolor A3(2). PMID : 16909271 : DOI : 10.1007/s00203-006-0158-9 Abstract >>
By using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor A3(2) stress-response sigma factor sigmaH. The promoter directed expression of the sigJ gene encoding an extracytoplasmic function (ECF) sigma factor. S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system, and S. coelicolor A3(2) from various developmental stages identified an identical transcription start point in both strains, corresponding to the sigJp promoter. The sigJp promoter was induced during sporulation of aerial hyphae. The level of the transcript from sigJp was dramatically reduced in a S. coelicolor A3(2) sigH mutant and unaffected in a sigF mutant. The S. coelicolor A3(2) core RNA polymerase, after complementation with sigmaH, was able to recognize the sigJp promoter in vitro. A sigJ mutation had no obvious effect on growth, stress response, differentiation, and production of antibiotics. The results suggested that the S. coelicolor A3(2) sigJ gene is under the control of stress-response sigmaH, thus indicating a cascade of sigma factors in Streptomyces stress response and development. Considering the expression of sigJ and its direct dependence upon developmentally-regulated sigmaH, we assume that sigmaJ may play a role in the later stages of development of S. coelicolor A3(2).
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35. |
Borovok I,
Gorovitz B,
Schreiber R,
Aharonowitz Y,
Cohen G,
( 2006 ) Coenzyme B12 controls transcription of the Streptomyces class Ia ribonucleotide reductase nrdABS operon via a riboswitch mechanism. PMID : 16547038 : DOI : 10.1128/JB.188.7.2512-2520.2006 PMC : PMC1428431 Abstract >>
Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5'-untranslated region of nrdABS contains a 123-nucleotide B12 riboswitch. Similar B12 riboswitches are present in the corresponding regions of eight other S. coelicolor genes. The effect of B12 on growth and nrdABS transcription was examined in a mutant in which the nrdJ gene, encoding the class II RNR, was deleted. B12 concentrations of just 1 mug/liter completely inhibited growth of the NrdJ mutant strain. Likewise, B12 significantly reduced nrdABS transcription. To further explore the mechanism of B12 repression, we isolated in the nrdJ deletion strain mutants that are insensitive to B12 inhibition of growth. Two classes of mutations were found to map to the B12 riboswitch. Both conferred resistance to B12 inhibition of nrdABS transcription and are likely to affect B12 binding. These results establish that B12 regulates overall RNR expression in reciprocal ways, by riboswitch regulation of the class Ia RNR nrdABS genes and by serving as a cofactor for the class II RNR.
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36. |
McCue LA,
Kwak J,
Babcock MJ,
Kendrick KE,
( 1992 ) Molecular analysis of sporulation in Streptomyces griseus. PMID : 1612433 : DOI : 10.1016/0378-1119(92)90556-5 Abstract >>
Previous evidence suggested that orf1590 from Streptomyces griseus has the potential to encode two polypeptide products from temporally regulated nested open frames (orfs) and that the longer polypeptide may be a DNA-binding protein. We have developed a hypothetical model of the role of orf1590 in sporulation of S. griseus and have begun to test this model by determining the nucleotide sequence of the orf1590 counterpart from Streptomyces coelicolor. The conservation of the helix-turn-helix domain and the two potential translation start codons is consistent with our model. Continued analysis of bald mutants of S. griseus has indicated that several prematurely synthesize sporulation septa and spore walls. One of these nonsporulating strains appears to be a bldA mutant of S. griseus. Complementation analysis suggests that at least three genetic loci are involved in the correct timing of deposition of sporulation septa and wall thickening.
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37. |
Fernández-Moreno MA,
Martín-Triana AJ,
Martínez E,
Niemi J,
Kieser HM,
Hopwood DA,
Malpartida F,
( 1992 ) abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor. PMID : 1569025 : DOI : 10.1128/jb.174.9.2958-2967.1992 PMC : PMC205950 Abstract >>
Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.
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38. |
Diacovich L,
Mitchell DL,
Pham H,
Gago G,
Melgar MM,
Khosla C,
Gramajo H,
Tsai SC,
( 2004 ) Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. PMID : 15518551 : DOI : 10.1021/bi049065v Abstract >>
Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.
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39. |
Fernández-Moreno MA,
Martínez E,
Boto L,
Hopwood DA,
Malpartida F,
( 1992 ) Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. PMID : 1527048 : Abstract >>
A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).
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40. |
Kim BJ,
Kim CJ,
Chun J,
Koh YH,
Lee SH,
Hyun JW,
Cha CY,
Kook YH,
( 2004 ) Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences. PMID : 15023980 : DOI : 10.1099/ijs.0.02941-0 Abstract >>
The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.
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41. |
Mimura H,
Nakanishi Y,
Hirono M,
Maeshima M,
( 2004 ) Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis. PMID : 15187077 : DOI : 10.1074/jbc.M406264200 Abstract >>
The H+-translocating pyrophosphatase (H+-PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H+-PPase expressed in Escherichia coli using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N- and C-terminal segments. Thirty-six of the mutant enzymes retained both pyrophosphatase and H+-translocating activities. Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S. coelicolor H+-PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus. We propose that the basic structure of H+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.
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42. |
Natsume R,
Ohnishi Y,
Senda T,
Horinouchi S,
( 2004 ) Crystal structure of a gamma-butyrolactone autoregulator receptor protein in Streptomyces coelicolor A3(2). PMID : 14757054 : DOI : 10.1016/j.jmb.2003.12.040 Abstract >>
The gamma-butyrolactone-type autoregulator/receptor systems in the Gram-positive bacterial genus Streptomyces regulate morphological differentiation or antibiotic production, or both. The autoregulator receptors act as DNA-binding proteins, and on binding their cognate ligands (gamma-butyrolactones) they are released from the DNA, thus serving as repressors. The crystal structure of CprB in Streptomyces coelicolor A3(2), a homologue of the A-factor-receptor protein, ArpA, in Streptomyces griseus, was determined. The overall structure of CprB shows that the gamma-butyrolactone receptors belong to the TetR family. CprB is composed of two domains, a DNA-binding domain and a regulatory domain. The regulatory domain contains a hydrophobic cavity, which probably serves as a ligand-binding pocket. On the basis of the crystal structure of CprB and on the analogy of the characteristics of ligand-TetR binding, the binding of gamma-butyrolactones to the regulatory domain of the receptors is supposed to induce the relocation of the DNA-binding domain through conformational changes of residues located between the ligand-binding site and the DNA-binding domain, which would result in the dissociation of the receptors from their target DNA.
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43. |
Taguchi S,
Kojima S,
Kumagai I,
Ogawara H,
Miura K,
Momose H,
( 1992 ) Isolation and partial characterization of SSI-like protease inhibitors from Streptomyces. PMID : 1490613 : DOI : 10.1016/0378-1097(92)90043-n Abstract >>
We attempted to screen a series of Streptomyces subtilisin inhibitor-like (SIL) proteins among several Streptomyces strains by using a highly sensitive assay system established by us. Of six randomly tested strains, four were found to produce SIL inhibitors as their major secreted proteins, suggesting that they might be distributed in a high frequency among this genus. Three inhibitors exhibited inhibition of both subtilisin BPN' and trypsin. Comparison of the amino terminal sequences of these isolated proteins with those of other reported SIL inhibitors revealed that the beta 1- and beta 2-sheets in SSI were highly conserved.
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44. |
Limauro D,
Avitabile A,
Puglia AM,
Bruni CB,
( 1992 ) Further characterization of the histidine gene cluster of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis of hisD. PMID : 1488552 : Abstract >>
We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine. A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced. Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products. Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank. S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.
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45. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
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46. |
Arabolaza A,
Shillito ME,
Lin TW,
Diacovich L,
Melgar M,
Pham H,
Amick D,
Gramajo H,
Tsai SC,
( 2010 ) Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor. PMID : 20690600 : DOI : 10.1021/bi1005305 PMC : PMC2927733 Abstract >>
The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.
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47. |
P?osko? E,
Arthur CJ,
Kanari AL,
Wattana-amorn P,
Williams C,
Crosby J,
Simpson TJ,
Willis CL,
Crump MP,
( 2010 ) Recognition of intermediate functionality by acyl carrier protein over a complete cycle of fatty acid biosynthesis. PMID : 20659690 : DOI : 10.1016/j.chembiol.2010.05.024 Abstract >>
It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis.
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48. |
Laskaris P,
Tolba S,
Calvo-Bado L,
Wellington EM,
Wellington L,
( 2010 ) Coevolution of antibiotic production and counter-resistance in soil bacteria. PMID : 20067498 : DOI : 10.1111/j.1462-2920.2009.02125.x Abstract >>
We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.
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49. |
Arthur CJ,
Williams C,
Pottage K,
P?osko? E,
Findlow SC,
Burston SG,
Simpson TJ,
Crump MP,
Crosby J,
( 2009 ) Structure and malonyl CoA-ACP transacylase binding of streptomyces coelicolor fatty acid synthase acyl carrier protein. PMID : 19555075 : DOI : 10.1021/cb900099e Abstract >>
Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.
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50. |
Fernández-Moreno MA,
Caballero JL,
Hopwood DA,
Malpartida F,
( 1991 ) The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. PMID : 1878971 : DOI : 10.1016/0092-8674(91)90120-n Abstract >>
The actII region, flanked by biosynthetic genes in the 25 kb act cluster of S. coelicolor, consists of four open reading frames, including a transcriptional activator for the biosynthetic genes, and genes controlling antibiotic export. A TTA codon (extremely rare in Streptomyces) is present both in actII-ORF2 (encoding a putative transmembrane export protein) and actII-ORF4 (the transcriptional activator gene). Change of the TTA in ORF4 to TTG reverses the normal interruption of actinorhodin synthesis caused by mutation in the pleiotropic regulatory gene bldA (which encodes the cell's tRNA(Leu)(UUA)). We conclude that initiation of actinorhodin synthesis via the actII-ORF4 product, and the final step in production, antibiotic export, are twin targets via which bldA exerts developmental control of actinorhodin production.
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51. |
Shiina T,
Tanaka K,
Takahashi H,
( 1991 ) Sequence of hrdB, an essential gene encoding sigma-like transcription factor of Streptomyces coelicolor A3(2): homology to principal sigma factors. PMID : 1840545 : DOI : 10.1016/0378-1119(91)90308-x Abstract >>
The complete nucleotide sequence of the hrdB gene, an essential gene of Streptomyces coelicolor A3(2), indicates the presence of an open reading frame encoding a putative polypeptide of 442 amino acid (aa) residues with an Mr of 48,412. The principal sigma-like transcriptional factor of S. coelicolor (HrdB) protein showed an extensive aa sequence homology with the known principal sigma factors of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Myxococcus xanthus. The degree of sequence similarity between HrdB protein and the known principal sigma factors was distinct from that observed between the principal sigma factors and the alternative (minor) sigma factors. Essentially all of the functional domains proposed for the principal sigma factor of E. coli were conserved in HrdB protein. The putative sigma factor, HrdB, like that of B. subtilis had a short internal nonconserved region, which might be characteristic of Gram+ species.
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52. |
Willems AR,
Tahlan K,
Taguchi T,
Zhang K,
Lee ZZ,
Ichinose K,
Junop MS,
Nodwell JR,
( 2008 ) Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA. PMID : 18207163 : DOI : 10.1016/j.jmb.2007.12.061 Abstract >>
Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism.
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53. |
Neal RJ,
Chater KF,
( 1987 ) Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. PMID : 2828187 : DOI : 10.1016/0378-1119(87)90378-7 Abstract >>
Previous studies had localised the gene (mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein (Mr 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene (tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.
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54. |
Bruton CJ,
Chater KF,
( 1987 ) Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2). PMID : 2821490 : DOI : 10.1093/nar/15.17.7053 PMC : PMC306192 Abstract >>
The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.
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55. |
Bhukya H,
Bhujbalrao R,
Bitra A,
Anand R,
( 2014 ) Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2). PMID : 25092919 : DOI : 10.1093/nar/gku587 PMC : PMC4150764 Abstract >>
Antibiotic production and resistance pathways in Streptomyces are dictated by the interplay of transcriptional regulatory proteins that trigger downstream responses via binding to small diffusible molecules. To decipher the mode of DNA binding and the associated allosteric mechanism in the sub-class of transcription factors that are induced by �^-butyrolactones, we present the crystal structure of CprB in complex with the consensus DNA element to a resolution of 3.25 ?. Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove. The crystal structure revealed that, CprB is bound to DNA as a dimer of dimers with the mode of binding being analogous to the broad spectrum multidrug transporter protein QacR from the antibiotic resistant strain Staphylococcus aureus. It was demonstrated that the CprB displays a cooperative mode of DNA binding, following a clamp and click model. Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator. Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the �^-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).
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56. |
Chater KF,
Bruton CJ,
Plaskitt KA,
Buttner MJ,
Méndez C,
Helmann JD,
( 1989 ) The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis. PMID : 2507166 : DOI : 10.1016/0092-8674(89)90876-3 Abstract >>
In the mycelial prokaryote S. coelicolor, whiG is a gene dispensable for growth but needed for the earliest stages of spore formation in aerial hyphae. Nucleotide sequencing indicates that whiG encodes an RNA polymerase sigma factor highly similar to the motility sigma factor (sigma 28) of B. subtilis. High copy number of an intact whiG gene caused sporulation in vegetative hyphae that are usually fated to lyse without sporulating. However, the introduction of many copies of a sigma 28-dependent promoter from B. subtilis into S. coelicolor reduced sporulation, suggesting partial sequestration of the whiG gene product by the foreign promoter sequences. We propose that the level of whiG sigma factor is crucial in determining the developmental fate of hyphae.
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57. |
Zhang R,
Xia H,
Xu Q,
Dang F,
Qin Z,
( 2013 ) Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2). PMID : 23710588 : DOI : 10.1111/1574-6968.12183 Abstract >>
The model organism Streptomyces coelicolor A3(2) harbors a 356-kb linear plasmid, SCP1. We report here development of a recombinational cloning method for deleting large segment from one telomere of SCP1 followed by replacing with the telomere of pSLA2 and sequentially inserting with the overlapping cosmids in vivo. The procedure depends on homologous recombination coupled with cleavage at telomere termini by telomere terminal protein. Using this procedure, we cloned the 81-kb avermectin and the 76-kb spinosad biosynthetic gene clusters into SCP1. Heterologous expression of avermectin production in S. coelicolor was detected. These results demonstrate the utility of SCP1 for cloning large DNA segments such as antibiotic biosynthetic gene clusters.
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58. |
Limauro D,
Avitabile A,
Cappellano C,
Puglia AM,
Bruni CB,
( 1990 ) Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2). PMID : 2199329 : DOI : 10.1016/0378-1119(90)90436-u Abstract >>
Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.
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59. |
( 1997 ) Isolation and expression of the catA gene encoding the major vegetative catalase in Streptomyces coelicolor M?ller. PMID : 9190825 : DOI : 10.1128/jb.179.12.4049-4052.1997 PMC : PMC179218 Abstract >>
We isolated the catA gene for the major vegetative catalase from Streptomyces coelicolor M?ller. It encodes a polypeptide of 488 residues (55,440 Da) that is highly homologous to typical monofunctional catalases. We investigated catA expression by analyzing both catA mRNA and catalase activity. catA expression was increased by H2O2 treatment but did not increase during stationary phase. A putative catalase (CatB) cross-reactive with anti-CatA antibody appeared during stationary phase and in the aerial mycelium.
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60. |
( 1997 ) The malEFG gene cluster of Streptomyces coelicolor A3(2): characterization, disruption and transcriptional analysis. PMID : 9197422 : DOI : 10.1007/s004380050458 Abstract >>
The malEFG gene cluster of the Gram-positive mycelial actinomycete Streptomyces coelicolor A3(2) was cloned and sequenced. MalEFG show only limited similarity to homologues involved in maltose and maltodextrin transport in other bacteria. Disruption of malE prevented the utilization of maltose as carbon source. Transcription of malE was induced by maltose and repressed by glucose.
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61. |
( 1996 ) Propionyl-CoA carboxylase from Streptomyces coelicolor A3(2): cloning of the gene encoding the biotin-containing subunit. PMID : 8868440 : DOI : 10.1099/13500872-142-3-649 Abstract >>
In Streptomyces coelicolor A3(2), polyketides are made from malonyl-CoA, which is presumed to be derived from acetyl-CoA by the action of acetyl-CoA carboxylase (ACC). No ACC activity was found in cell-free extracts of S. coelicolor. However, propionyl-CoA carboxylase (PCC) activity was detected at substantial levels. Fixation of CO2 by ACC and PCC occurs by covalent bonding of CO2 to a biotin-containing protein. Most bacteria have a single small biotinylated protein of approximately 22 kDa, but S. coelicolor contains three larger biotin-containing proteins (approximately 145, 88 and 70 kDa). To determine which biotinylated protein was associated with PCC activity, the enzyme was purified and shown to comprise an alpha subunit (biotin-containing) of 88 kDa and a beta subunit of 66 kDa. The N-terminal sequences of these proteins were determined and, using an oligonucleotide probe, the gene for the alpha subunit (pccA) was cloned.
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62. |
( 1996 ) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. PMID : 8843436 : DOI : 10.1046/j.1365-2958.1996.6191336.x Abstract >>
A Supercos-1 library carrying chromosomal DNA of a plasmid-free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization. The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids. The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx. 12.5 kb. More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia. Genes could be cloned by direct transformation and complementation of S. coelicolor mutants with cosmids isolated from Escherichia coli, selecting for insertion into the chromosome by homologous recombination. As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats.
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63. |
( 1996 ) Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein. PMID : 8824610 : DOI : 10.1128/jb.178.19.5660-5667.1996 PMC : PMC178404 Abstract >>
We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.
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64. |
( 1996 ) Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2). PMID : 8752351 : DOI : 10.1128/jb.178.17.5295-5301.1996 PMC : PMC178330 Abstract >>
We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.
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65. |
( 1997 ) Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes. PMID : 9044287 : DOI : 10.1046/j.1365-2958.1997.d01-1878.x Abstract >>
malR of Streptomyces coelicolor A3(2) encodes a homologue of the Lacl/GalR family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP-dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose-insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. malR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translation start codon.
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66. |
( 1993 ) Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete. PMID : 8328954 : DOI : 10.1042/bj2930131 PMC : PMC1134330 Abstract >>
Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.
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67. |
( 1995 ) Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2). PMID : 8596463 : DOI : 10.1111/j.1365-2958.1995.mmi_18010089.x Abstract >>
In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgBl led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.
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68. |
( 1993 ) Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans. PMID : 8387993 : DOI : 10.1128/jb.175.10.3067-3074.1993 PMC : PMC204627 Abstract >>
SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.
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69. |
( 1996 ) Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system. PMID : 8655502 : DOI : 10.1128/jb.178.11.3221-3231.1996 PMC : PMC178074 Abstract >>
Streptomycete antibiotic synthesis is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. The S. coelicolor absA locus was defined by four UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. We show that the absA locus encodes a putative eubacterial two-component sensor kinase-response regulator system. All four mutations lie within a single open reading frame, designated absA1, which is predicted to encode a sensor histidine kinase. A second gene downstream of absA1, absA2, is predicted to encode the cognate response regulator. In marked contrast to the antibiotic-deficient phenotype of the previously described absA mutants, the phenotype caused by disruption mutations in the absA locus is precocious hyperproduction of the antibiotics actinorhodin and undecylprodigiosin. Precocious hyperproduction of these antibiotics is correlated with premature expression of XylE activity in a transcriptional fusion to an actinorhodin biosynthetic gene. We propose that the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor.
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70. |
( 1996 ) Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor. PMID : 8550471 : DOI : 10.1128/jb.178.2.490-495.1996 PMC : PMC177683 Abstract >>
A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic aldehyde dehydrogenases that utilize NAD+, particularly to the MmsA protein from P. aeruginosa. No significant similarity was found between the deduced product of ORF1 and known proteins in the databases. An S. coelicolor msdA mutant, constructed by insertion of a hygromycin resistance gene (hyg) into the msdA coding region, lost the MSDH activity and the ability to grow in a minimal medium with valine or isobutyrate as the sole carbon source but grew on propionate. The msdA::hyg mutation was complemented by introduction of the msdA gene on a plasmid. When the S. coelicolor msdA gene was overexpressed in Escherichia coli under the control of the T7 promoter, a protein of 51-kDa, corresponding to the approximate mass of the predicted S. coelicolor msdA product (52.6 kDa), and specific MSDH activity were detected. These results strongly suggest that msdA indeed encodes the MSDH that is involved in valine catabolism in S. coelicolor.
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71. |
( 1994 ) Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase. PMID : 8063104 : DOI : 10.1016/0378-1119(94)90832-x Abstract >>
A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membrane-associated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S. lividans. The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs). Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK. The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues. Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production. This result implies the involvement of afsK in the regulation of secondary metabolism. The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK-disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR. Southern hybridization experiments showed that nt sequences homologous to afsK, as well as afsR, were distributed among many Streptomyces spp. It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces.
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72. |
( 1994 ) Comparative studies on the primary structures and inhibitory properties of subtilisin-trypsin inhibitors from Streptomyces. PMID : 8143745 : DOI : 10.1111/j.1432-1033.1994.tb18694.x Abstract >>
Three novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor-like (SIL) inhibitors were isolated from culture supernatant of Streptomyces; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SIL4 from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg-Glu for SIL2 and SIL3, and Lys-Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor-subtilisin complex under acidic conditions. The presence of an arginine/lysine residue at the P1 site was in agreement with their trypsin-inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in beta-sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (Ki) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.
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73. |
Puttikhunt C,
Nihira T,
Yamada Y,
( 1995 ) Cloning, nucleotide sequence, and transcriptional analysis of the nusG gene of Streptomyces coelicolor A3(2), which encodes a putative transcriptional antiterminator. PMID : 7715599 : DOI : 10.1007/bf00425829 Abstract >>
A 3 kb genomic fragment containing the nusG gene of Streptomyces coelicolor A3(2) was identified, cloned and sequenced. Sequence analysis revealed 3 complete and 2 truncated open reading frames (ORFs): truncated ORFU (similar to a Bacillus gene encoding a thermostable aspartate aminotransferase)-secE (94 amino acids; 79.0% similarity to Escherichia coli SecE)-nusG (300 amino acids; 73.3% similarity to E. coli NusG)-rplK (144 amino acids; 88.5% similarity to E. coli ribosomal subunit L11)-truncated rplA (similar to E. coli ribosomal subunit L1). The gene organization secE-nusG-rplKA exactly matches that in E. coli. Transcriptional analyses by the primer extension method revealed one transcriptional start site each for secE and nusG, and two sites for rplK. The presence of promoters was also confirmed with the aid of a promoter-probe vector.
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74. |
McCormick JR,
Su EP,
Driks A,
Losick R,
( 1994 ) Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ. PMID : 7830569 : DOI : 10.1111/j.1365-2958.1994.tb01285.x Abstract >>
A homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor. The gene was located on the physical map of the chromosome at about '11 o'clock' (in the vicinity of glkA, hisA and trpB). Surprisingly, a null mutant in which the 399-codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation. This indicates that cell division may not be essential for the growth and viability of S. coelicolor. The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores.
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75. |
Calcutt MJ,
( 1994 ) Gene organization in the dnaA-gyrA region of the Streptomyces coelicolor chromosome. PMID : 7828880 : DOI : 10.1016/0378-1119(94)90628-9 Abstract >>
The gene organization has been determined for an 8-kb portion of the Streptomyces coelicolor chromosome close to the origin of DNA replication (oriC). Hybridization and DNA sequence analyses revealed the presence of five open reading frames (ORFs) oriented in the same direction as the proximal dnaA and dnaN genes. The deduced products of three of the ORFs have been identified as the S. coelicolor homologs of RecF, GyrB and GyrA. These genes are usually clustered in the dnaA region of bacterial chromosomes. In S. coelicolor however, the usual gene arrangement is altered. The recF gene is flanked by two ORFs, one of which encodes a protein with significant similarity to 6-phosphogluconate dehydrogenases (6PGDH), an enzyme that is not immediately linked to DNA metabolism.
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76. |
( 1994 ) DNA sequence and functions of the actVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2). PMID : 7929165 : Abstract >>
Six open reading frames (ORFs) were identified by DNA sequencing of 5.7 kilobase pairs at the left end of the act cluster (the so-called "actVI region"), in the order: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. ORF1-4 are transcribed rightward and in the same direction as the ORFs of the actVA region which lies to the right of the actVI region, whereas ORFA and ORFB run in the opposite direction. By complementation of mutants and gene disruption of the wild type strain, the two previously genetically characterized actVI mutations were assigned to ORF1. Although disruption of ORFB and ORF4, using phi C31 derivatives, did not cause any obvious change in actinorhodin production, defects in actinorhodin synthesis were obtained by insertional inactivation of ORFA, ORF1, ORF2, or ORF3. RNA analysis within the ORF1/ORFA intergenic region showed overlapping divergent promoters, at least one of which is under the control of the actII-ORF4 gene product, the transcriptional activator of the act cluster. Data base searches with the deduced products of ORFB and ORF3 failed to show any significant similarities with other known proteins. The deduced product of ORFA strongly resembles those of genes of unknown function from Saccharopolyspora hirsuta and Streptomyces roseofulvus, located within polyketide synthase clusters. The ORF1 product strongly resembles beta-hydroxyacyl-CoA dehydrogenases of bacteria and mammals and the ORF2 and ORF4 products resemble each other and enoyl reductases from bacteria, animals, and plants, with a highly conserved cofactor-binding domain. These findings strongly suggest that the actVI region is involved in catalyzing reduction processes that determine the two stereochemical configurations at C-3/C-15 during actinorhodin biosynthesis. A scheme is proposed for the middle steps of the biosynthesis, that is formation of the pyran ring, leading to the benzoisochromanequinone structure.
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77. |
Tan H,
Chater KF,
( 1993 ) Two developmentally controlled promoters of Streptomyces coelicolor A3(2) that resemble the major class of motility-related promoters in other bacteria. PMID : 7679386 : DOI : 10.1128/jb.175.4.933-940.1993 PMC : PMC193004 Abstract >>
Experiments were designed to allow isolation of Streptomyces coelicolor promoters that depend on the whiG sporulation gene, which encodes a putative sigma factor important in the sporulation of aerial hyphae. The strategy, based on earlier evidence that sigma WhiG is limiting for sporulation (K. F. Chater, C. J. Burton, K. A. Plaskitt, M. J. Buttner, C. M?ndez, and J. Helmann, Cell 59:133-143, 1989) was to seek DNA fragments that inhibit sporulation in aerial hyphae when present at a high copy number. In a suitable Sau3AI-generated library of DNA from S. coelicolor A3(2), two inserts were found to inhibit sporulation. Both inserts caused expression of the adjacent xylE reporter gene present in the vector in a developmentally normal strain of S. coelicolor, but there was no xylE expression in an otherwise isogenic whiG mutant. S1 nuclease protection experiments were done with RNAs isolated from these plasmid-bearing strains or from the wild-type strain lacking either recombinant plasmid. In each case, an apparent transcription start site was found upstream of an apparent open reading frame (ORF) and just downstream of sequences that resemble consensus features of promoters for motility-related genes in Bacillus subtilis and coliform bacteria. Such promoters depend on sigma factors (sigma D and sigma F, respectively) particularly similar to the deduced whiG gene product. Each of the putative whiG-dependent promoters is within an ORF that is upstream of, and potentially translationally coupled to, the putative whiG-dependent ORF (although use of one of the promoters would necessitate the use of a different start codon, further downstream). Thus, in unknown circumstances, the whiG-dependent ORFs may be expressed from a more remote promoter as part of a complex transcription unit.
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78. |
( 1993 ) Analysis of the transfer region of the Streptomyces plasmid SCP2. PMID : 7968512 : DOI : 10.1111/j.1365-2958.1993.tb00912.x Abstract >>
pIJ903, a bifunctional derivative of the 31.4 kb low-copy-number, conjugative Streptomyces plasmid SCP2*, was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.
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79. |
Bedford DJ,
Laity C,
Buttner MJ,
( 1995 ) Two genes involved in the phase-variable phi C31 resistance mechanism of Streptomyces coelicolor A3(2). PMID : 7642495 : DOI : 10.1128/jb.177.16.4681-4689.1995 PMC : PMC177233 Abstract >>
The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to phi C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl+ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M(r)S of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.
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80. |
Taguchi S,
Kikuchi H,
Kojima S,
Kumagai I,
Nakase T,
Miura K,
Momose H,
( 1993 ) High frequency of SSI-like protease inhibitors among Streptomyces. PMID : 7763545 : Abstract >>
N/A
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81. |
Revill WP,
Bibb MJ,
Hopwood DA,
( 1995 ) Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant. PMID : 7608065 : DOI : 10.1128/jb.177.14.3946-3952.1995 PMC : PMC177122 Abstract >>
Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.
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82. |
Chater KF,
Bruton CJ,
( 1985 ) Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. PMID : 2992952 : PMC : PMC554433 Abstract >>
At least 17 kb of DNA from the large unisolatable Streptomyces coelicolor A3(2) plasmid SCP1 are concerned with methylenomycin biosynthesis. Mutational cloning analysis, using insert-directed integration of att site deleted phage vectors into an SCP1-containing host, provided evidence of two large transcription units, of at least 6.6 kb and 9.5 kb. At the leftmost apparent end of the larger (left-hand) transcription unit is a region apparently involved in negative regulation of methylenomycin biosynthesis: when fragments from this region were used to direct phage integration, marked overproduction of methylenomycin resulted. The methylenomycin resistance determinant is located at the rightmost end of this same transcription unit. Hybridisation analysis with 13 kb of the cloned mmy region showed that it was closely similar to a segment of pSV1, a plasmid that specifies methylenomycin biosynthesis in S. violaceus-ruber SANK 95570.
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83. |
Kendall K,
Cullum J,
( 1986 ) Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2). PMID : 3010046 : DOI : 10.1007/bf00331643 Abstract >>
We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA. A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose.
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84. |
Wray LV,
Fisher SH,
( 1988 ) Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase. PMID : 2906310 : DOI : 10.1016/0378-1119(88)90041-8 Abstract >>
The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.
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85. |
( 1998 ) Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis. PMID : 9643542 : DOI : 10.1046/j.1365-2958.1998.00832.x Abstract >>
Nucleotide sequences homologous to arpA encoding the A-factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB, each encoding an ArpA-like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage-derived single-stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low-copy-number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA/A-factor system in Streptomyces griseus.
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86. |
( 1998 ) A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2). PMID : 9701826 : DOI : 10.1046/j.1365-2958.1998.00939.x Abstract >>
whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream on hrdB, which encodes the principal sigma factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.
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87. |
( 1998 ) A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. PMID : 9534242 : DOI : 10.1099/00221287-144-3-727 Abstract >>
In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.
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88. |
( 1997 ) Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex. PMID : 9418255 : DOI : 10.1111/j.1574-6968.1997.tb12773.x Abstract >>
An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.
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89. |
( 1998 ) Analysis of a Streptomyces coelicolor A3(2) locus containing the nucleoside diphosphate kinase (ndk) and folylpolyglutamate synthetase (folC) genes. PMID : 9503623 : DOI : 10.1111/j.1574-6968.1998.tb12873.x Abstract >>
A 3.6-kb DNA fragment from Streptomyces coelicolor A3(2) with the genes valS probably encoding a valyl-tRNA synthetase, folC encoding folylpolyglutamate synthetase, and ndk encoding a nucleoside diphosphate kinase was analysed. folC and ndk are separated by a small open reading frame of unknown function, orfX. The deduced folC gene product is a protein of 46,677 Da whose sequence is similar to other folylpolyglutamate synthetases and folylpolyglutamate synthetase-dihydrofolate synthetases from both Gram-positive and Gram-negative bacteria. After cloning folC behind the lacZ promoter, the Streptomyces folC complemented a folC mutant of Escherichia coli. An essential function for Streptomyces folC was suggested by the fact that it could not be mutated using a conventional gene disruption technique.
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90. |
( 1998 ) The bldD gene of Streptomyces coelicolor A3(2): a regulatory gene involved in morphogenesis and antibiotic production. PMID : 9515925 : PMC : PMC107056 Abstract >>
The bld mutants of Streptomyces coelicolor A3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type S. coelicolor DNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known bldD mutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated bldD, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the bldD-containing fragment amplified from the chromosome of a bldD mutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of bldD transcript levels in the bldD+ and bldD mutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of bldD transcripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the bldD start codon and 7 nucleotides downstream from -10 and -35 sequences resembling E. coli-like streptomycete promoters.
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91. |
( 1997 ) Cloning, sequencing and transcriptional analysis of a Streptomyces coelicolor operon containing the rplM and rpsI genes encoding ribosomal proteins ScoL13 and ScoS9. PMID : 9439573 : DOI : 10.1007/s004380050627 Abstract >>
The N-terminal amino acid sequences of two peptides derived from a Streptomyces coelicolor ribosomal protein were determined and degenerate oligonucleotide primers derived from these sequences were used as probes for the screening of a chromosomal DNA library of S. coelicolor. Two positive clones were isolated and DNA sequencing of a 1740-bp region of these clones that hybridised with the probes revealed the presence of four genes, two of them incomplete. The deduced products of the two complete genes. rplM and rpsI, showed clear similarities to L13 and S9 ribosomal proteins from various organisms. Promoter-probe and primer extension experiments suggest that the two genes form a single transcriptional unit. The specific rate of synthesis of both proteins was high at early stages of growth but decreased later.
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( 1998 ) Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor M?ller. PMID : 9466266 : DOI : 10.1046/j.1365-2958.1998.00674.x Abstract >>
A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp. The gene for NiSOD (sodN) was cloned from S. coelicolor M?ller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E. coli, indicating that N-terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.
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( 1997 ) A glgC gene essential only for the first of two spatially distinct phases of glycogen synthesis in Streptomyces coelicolor A3(2). PMID : 9401038 : DOI : 10.1128/jb.179.24.7784-7789.1997 PMC : PMC179742 Abstract >>
By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.
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