| 1. |
Yonekawa T,
Ohnishi Y,
Horinouchi S,
( 2001 ) A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens. PMID : 11272820 : DOI : 10.1271/bbb.65.156 Abstract >>
A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.
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2. |
Martínez E,
Culebras E,
( 1999 ) Cloning and characterization of a regulatory gene of the SARP family and its flanking region from Streptomyces ambofaciens. PMID : 10628855 : DOI : 10.1007/s004380051135 Abstract >>
In a search for activators of secondary metabolism we isolated a 12.6-kb DNA fragment from a genomic library of Streptomyces ambofaciens NRRL 2240 (the spiramycin producer). Sequencing of 6 kb of the cloned fragment revealed a cluster of four ORFs (ORF1-4) whose deduced products showed similarities to those of other genes involved in polyketide biosynthesis, including a pathway-specific regulatory gene of the SARP family. The results of insertional inactivation of some of the cloned genes clearly indicate that the isolated cluster does not code for spiramycin production, suggesting that some other polyketide compound might well be produced by this strain.
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3. |
Choulet F,
Gallois A,
Aigle B,
Mangenot S,
Gerbaud C,
Truong C,
Francou FX,
Borges F,
Fourrier C,
Guérineau M,
Decaris B,
Barbe V,
Pernodet JL,
Leblond P,
( 2006 ) Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens. PMID : 16952952 : DOI : 10.1128/JB.00734-06 PMC : PMC1595491 Abstract >>
The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.
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4. |
Choulet F,
Aigle B,
Gallois A,
Mangenot S,
Gerbaud C,
Truong C,
Francou FX,
Fourrier C,
Guérineau M,
Decaris B,
Barbe V,
Pernodet JL,
Leblond P,
( 2006 ) Evolution of the terminal regions of the Streptomyces linear chromosome. PMID : 16956972 : DOI : 10.1093/molbev/msl108 Abstract >>
Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities.
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5. |
Catakli S,
Andrieux A,
Decaris B,
Dary A,
( 2005 ) Sigma factor WhiG and its regulation constitute a target of a mutational phenomenon occurring during aerial mycelium growth in Streptomyces ambofaciens ATCC23877. PMID : 15808936 : DOI : 10.1016/j.resmic.2004.12.001 Abstract >>
The genetic instability of Streptomyces ambofaciens affects the pigmentation of colonies and generates a variety of mutants the majority of which display large genome rearrangements. Among them, the Pig-pap mutants, which probably result from a mutational event occurring during aerial mycelium growth, display specific features, since they are unable to sporulate and do not harbor any large detectable genome rearrangements. To identify the mutational event causing their phenotype, three Pig-pap mutants originating from three independent mutational events were characterized. These mutants exhibited a whiG-like phenotype which was suppressed by the introduction of one copy of Streptomyces coelicolor whiG. Their own whiG gene was devoid of mutations and appeared to be transcribed at a level similar to that of the WT. However, whiH, the expression of which depends on sigma(WhiG), was not transcribed in any of the three Pig-pap mutants, suggesting that the sigma(WhiG) was absent or inactive. This suggests that in these Pig-pap mutants, the regulation of sigma(WhiG) might be affected. Finally, the introduction of S. coelicolor whiG in one of these Pig-pap mutants restored not only pigmentation and sporulation, but also the ability to once again form white papillae. Analyses of transgene whiG in these papillae revealed that it constitutes a mutational target during aerial mycelium formation when integrated into the genome of this Pig-pap mutant.
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6. |
Yonekawa T,
Ohnishi Y,
Horinouchi S,
( 2005 ) A calmodulin-like protein in the bacterial genus Streptomyces. PMID : 15766784 : DOI : 10.1016/j.femsle.2005.02.003 Abstract >>
The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix-loop-helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its alpha-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant DeltacabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera.
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7. |
Schoner B,
Geistlich M,
Rosteck P,
Rao RN,
Seno E,
Reynolds P,
Cox K,
Burgett S,
Hershberger C,
( 1992 ) Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins. PMID : 1612454 : DOI : 10.1016/0378-1119(92)90545-z Abstract >>
The three macrolide-resistance-encoding genes, tlrC from Streptomyces fradiae, srmB from Streptomyces ambofaciens, and carA from Streptomyces thermotolerans, encode proteins that possess significant sequence similarity to ATP-dependent transport proteins. The N-terminal and C-terminal halves of these proteins are very similar to each other and contain highly conserved regions that resemble ATP-binding domains typically present within the superfamily of ATP-dependent transport proteins. These observations suggest that the mechanism by which these genes confer resistance to macrolides is due to export of the antibiotics, a process that is driven by energy derived from ATP hydrolysis.
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8. |
Simonet JM,
Schneider D,
Volff JN,
Dary A,
Decaris B,
( 1992 ) Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity. PMID : 1612449 : DOI : 10.1016/0378-1119(92)90539-2 Abstract >>
DNA amplification and deletions occur at high frequency in unstable regions localized on the Streptomyces ambofaciens chromosome. The structure of these regions was investigated, leading to the identification of internal reiterations which could play a role in the deletion and/or amplification mechanism(s). UV irradiation and treatments with mitomycin C, oxolinic acid and novobiocin were shown to efficiently induce genetic instability. Finally, mutator strains were isolated, in which genetic instability was dramatically increased. The involvement of an SOS-like response in genetic instability in S. ambofaciens is proposed.
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9. |
Kim H,
Kim SH,
Ying YH,
Kim HJ,
Koh YH,
Kim CJ,
Lee SH,
Cha CY,
Kook YH,
Kim BJ,
( 2005 ) Mechanism of natural rifampin resistance of Streptomyces spp. PMID : 16094866 : DOI : 10.1016/j.syapm.2005.02.009 Abstract >>
In a previous phylogenetic study of the genus Streptomyces using the rpoB gene, N531, which stands for an aspargine residue in position 531 of RpoB instead of serine (S531), known to be associated with natural rifampin resistance in several organisms, was also observed in the RpoB of several Streptomyces species. To determine whether N531 is associated with the rifampin resistance of Streptomyces strains, we analyzed the rifampin minimum inhibitory concentrations (MICs) of 11 strains of the N531 RpoB type (putative rifampin resistant strains) and of 12 strains of the S531 RpoB type. (putative rifampin susceptible strains). In general, the N531 RpoB types showed higher MIC levels (16-128 microg/ml) than the S531 RpoB types (0-8 microg/ml). To determine the isolation frequencies of N531 RpoB types versus rifampin concentration, we applied screening methods involving different rifampin concentrations (0, 20 and 100 microg/ml) to Korean soils. Higher isolation frequencies of the N531 RpoB types were observed at the higher rifampin concentrations. In addition, during the course of this study we developed an allele specific PCR method to detect rifampin resistant Streptomyces strains. Our results strongly suggested that N531 might be involved in a major mechanism of natural rifampin resistance in strains of the genus Streptomyces.
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10. |
Hsu JS,
Yang YB,
Deng CH,
Wei CL,
Liaw SH,
Tsai YC,
( 2004 ) Family shuffling of expandase genes to enhance substrate specificity for penicillin G. PMID : 15466573 : DOI : 10.1128/AEM.70.10.6257-6263.2004 PMC : PMC522083 Abstract >>
Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.
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11. |
Roth V,
Aigle B,
Bunet R,
Wenner T,
Fourrier C,
Decaris B,
Leblond P,
( 2004 ) Differential and cross-transcriptional control of duplicated genes encoding alternative sigma factors in Streptomyces ambofaciens. PMID : 15292136 : DOI : 10.1128/JB.186.16.5355-5365.2004 PMC : PMC490935 Abstract >>
The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named sigma(B(R)) and sigma(B(L))) belonging to the sigma(B) general stress response family in Bacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically in S. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. While hasL is monocistronic, hasR is arranged in a polycistronic unit with two upstream open reading frames, arsR and prsR, that encode putative anti-anti-sigma and anti-sigma factors, respectively. Transcription of each has gene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by the B. subtilis general stress response sigma factor sigma(B). Expression from this type of promoter for each of the has genes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate that hasR is 100 times more strongly expressed than hasL from the sigma(B)-like promoter. Transcription from the second promoter of each gene (located upstream of arsR in the case of the hasR locus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and double has mutant strains revealed that sigma(B(R)) and sigma(B(L)) direct their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the sigma factors implied in stress response in Streptomyces.
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12. |
Geistlich M,
Losick R,
Turner JR,
Rao RN,
( 1992 ) Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. PMID : 1508047 : DOI : 10.1111/j.1365-2958.1992.tb01374.x Abstract >>
A key step in the biosynthesis of macrolide antibiotics is the assembly of a large macrocyclic lactone ring by a multienzyme protein complex called the polyketide synthase. In the species Streptomyces ambofaciens, the polyketide synthase for the assembly of the 16-membered ring of the macrolide antibiotic spiramycin is encoded by the biosynthetic gene srmG. Here we show that the accumulation of transcripts from the srmG promoter is governed by the regulatory gene srmR, whose predicted product, a 65 kDa polypeptide, is not significantly similar in its deduced amino acid sequence to that of previously reported proteins in the protein databases. The srmR gene product is also required for the accumulation of transcripts from srmX, an additional gene in the vicinity of srmR, but not for the accumulation of transcripts from srmR itself. Interestingly, mutations in srmR prevent the accumulation of transcripts from the spiramycin resistance gene srmB, but this is an indirect consequence of the failure of srmR mutants to produce spiramycin, which is an inducer of its own resistance gene. The possibility that srmR is the prototype for a new class of regulatory genes governing early events in the biosynthesis of macrolide antibiotics is discussed.
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13. |
Pang X,
Aigle B,
Girardet JM,
Mangenot S,
Pernodet JL,
Decaris B,
Leblond P,
( 2004 ) Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome. PMID : 14742212 : DOI : 10.1128/aac.48.2.575-588.2004 PMC : PMC321545 Abstract >>
Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.
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14. |
Choi SU,
Kim MK,
Ha HS,
Hwang YI,
( 2008 ) In vivo functions of the gamma-butyrolactone autoregulator receptor in Streptomyces ambofaciens producing spiramycin. PMID : 18058070 : DOI : 10.1007/s10529-007-9613-1 Abstract >>
A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.
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15. |
Pan G,
Gao X,
Fan K,
Liu J,
Meng B,
Gao J,
Wang B,
Zhang C,
Han H,
Ai G,
Chen Y,
Wu D,
Liu ZJ,
Yang K,
( 2017 ) Structure and Function of a C-C Bond Cleaving Oxygenase in Atypical Angucycline Biosynthesis. PMID : 28103689 : DOI : 10.1021/acschembio.6b00621 Abstract >>
C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.
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16. |
Boccard F,
Smokvina T,
Pernodet JL,
Friedmann A,
Guérineau M,
( 1989 ) Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces. PMID : 2657820 : Abstract >>
pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.
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17. |
Boccard F,
Smokvina T,
Pernodet JL,
Friedmann A,
Guérineau M,
( 1989 ) The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. PMID : 2721504 : PMC : PMC400899 Abstract >>
Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.
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18. |
( 1996 ) An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes. PMID : 8885397 : DOI : 10.1099/13500872-142-10-2815 Abstract >>
Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0.93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.
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19. |
( 1996 ) New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces. PMID : 8597568 : DOI : 10.1016/0167-4838(95)00207-3 Abstract >>
Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.
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20. |
( 1993 ) Analysis of genome instability in Streptomyces ambofaciens. PMID : 8277241 : DOI : 10.1099/00221287-139-11-2559 Abstract >>
Genetic instability in Streptomyces ambofaciens DSM 40697 is correlated with genomic instability characterized by multiple rearrangements (deletions and/or amplifications) occurring in a large unstable region. We have focused on one of the two amplifiable DNA loci which were mapped in this region: the amplifiable unit of DNA locus 6 (AUD6). The nucleotide sequence of one AUD6 fragment of 1.9 kb reveals the presence of two open reading frames (ORF1 and ORF2) on the basis of the typical Streptomyces base composition at each of the three positions within codons. ORF1 shows some similarity with a gene encoding a regulatory protein. The presence of potential genes in this unstable locus was unexpected because deletions occurred with high frequency within this region in the genetic instability-derived mutant strains. However, transcription analyses by S1 nuclease protection experiments on the wild-type strain showed transcription of both ORF1 and ORF2. Moreover, the amplified strain reveals increased transcription of ORF1 but no transcription of ORF2. The amplification therefore results in a switch in transcription. The unstable region of S. ambofaciens DSM 40697 therefore is not a 'silent' region because at least some loci are transcribed.
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21. |
( 1995 ) Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens. PMID : 8559072 : DOI : 10.1111/j.1365-2958.1995.mmi_17030533.x Abstract >>
pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.
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22. |
( 1993 ) Primary structure analysis of a duplicated region in the amplifiable AUD6 locus of Streptomyces ambofaciens DSM40697. PMID : 8243982 : DOI : 10.1111/j.1574-6968.1993.tb06486.x Abstract >>
In Streptomyces ambofaciens, an amplifiable unit of DNA (AUD6) contains two homologous sequences, one located on the right extremity of the AUD (S1R), the other being internal (IHS). This paper presents the molecular analysis of this duplication. The nucleotide sequences are almost identical (95%) and each contains an ORF of about 330 codons, the two ORFs being nearly identical. The two hypothetical proteins, deduced from these sequences, show about 30% identity with different bacterial repressors. They also show a particularly strong similarity (90% identity between the full-length sequences) with hypothetical proteins of Streptomyces lividans 66 encoded by sequences also present on an amplifiable DNA region (AUD1).
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23. |
( 1993 ) Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer. PMID : 8366038 : DOI : 10.1128/jb.175.17.5529-5538.1993 PMC : PMC206609 Abstract >>
pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.
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24. |
( 1994 ) Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2. PMID : 8029324 : DOI : 10.1006/plas.1994.1018 Abstract >>
pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism. Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin). We report here the study of the second region. Its nucleotide sequence was determined and analysed for open reading frames (ORFs). Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa). orf183 is not necessary for replication. The function of orf50 is unknown. repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2. repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.
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25. |
( 1993 ) Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens. PMID : 7934842 : DOI : 10.1111/j.1365-2958.1993.tb00950.x Abstract >>
pSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single-stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling-circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M-O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.
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26. |
( 1994 ) Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae. PMID : 7928973 : DOI : 10.1128/jb.176.19.6107-6119.1994 PMC : PMC196831 Abstract >>
Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.1 and S. ambofaciens ATCC 15154, are approximately 80% identical over all 363 amino acids and 96% identical over a span of the first N-terminal 107 amino acids, respectively, to the deduced product of the Streptomyces coelicolor vdh gene. The organization of the regions flanking the vdh genes is the same in all three species. Inactivation of the genomic copy of the vdh gene in S. fradiae and S. ambofaciens by insertion of a hygromycin resistance (hyg) gene caused loss of the valine dehydrogenase (Vdh) activity, and thus only one enzyme is responsible for the Vdh activity in these organisms. Analysis of the culture broth by bioassay revealed that the vdh::hyg mutants produce an approximately sixfold-lower level of tylosin and an approximately fourfold-lower level of spiramycin than the wild-type S. fradiae and S. ambofaciens strains, while maintaining essentially identical growth in a defined minimal medium with either 25 mM ammonium ion or 0.05% asparagine as the nitrogen source. The addition of the valine catabolite, propionate or isobutyrate, and introduction of the wild-type vdh gene back to each vdh::hyg mutant reversed the negative effect of the vdh::hyg mutation on spiramycin and tylosin production. These data show that the catabolism of valine is a major source of fatty acid precursors for macrolide biosynthesis under defined growth conditions and imply that amino acid catabolism is a vital source of certain antibiotic precursors in actinomycetes.
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27. |
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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28. |
Wang W,
Li J,
Li H,
Fan K,
Liu Y,
( 2019 ) Crystal structure of AlpK: An essential monooxygenase involved in the biosynthesis of kinamycin. PMID : 30739782 : DOI : 10.1016/j.bbrc.2019.01.077 Abstract >>
AlpK is an essential monooxygenase involved in the biosynthesis of kinamycin. It catalyzes the C5-hyfroxylattion of the crucial benzo[b]-fluorence intermediate in kinamycin synthesis. However, the structure and mechanism of AlpK is unclear. Here, we report the first structure of AlpK in complex with FAD. Our structure sheds light on the catalytic mechanism of AlpK.
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29. |
( 1998 ) Replicase, excisionase, and integrase genes of the Streptomyces element pSAM2 constitute an operon positively regulated by the pra gene. PMID : 9620953 : PMC : PMC107804 Abstract >>
pSAM2 is a site-specific integrative element from Streptomyces ambofaciens. The pra gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the pra gene was placed under the control of the inducible tipAp promoter. Transformation of Streptomyces lividans by these derivatives was efficient only when pra expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the pra expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes repSA, xis, and int coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by pra.
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30. |
( 1998 ) Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens. PMID : 9826694 : DOI : 10.1073/pnas.95.24.14296 PMC : PMC24367 Abstract >>
The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.
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