( 2002 )
A novel streptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene.
PMID : 12115057 : DOI : 10.1007/s00203-002-0430-6
An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.
( 1975 )
PMID : 1134373 : DOI : 10.1016/0076-6879(75)43122-6
( 1999 )
Disruption of an aromatase/cyclase from the oxytetracycline gene cluster of Streptomyces rimosus results in production of novel polyketides with shorter chain lengths.
PMID : 10551844 :
Oxytetracycline is a polyketide antibiotic made by Streptomyces rimosus. From DNA sequencing, the gene product of otcD1 is deduced to function as a bifunctional cyclase/aromatase involved in ring closure of the polyketide backbone. Although otcD1 is contiguous with the ketoreductase gene, they are located an unusually large distance from the genes encoding the "minimal polyketide synthase" of the oxytetracycline gene cluster. A recombinant, disrupted in the genomic copy of otcD1, made four novel polyketides, all of shorter chain length (by up to 10 carbons) than oxytetracycline. All four novel structures contained the unusual carboxamido group, typical of oxytetracycline. This implies that the carboxamido group is present at the start of biosynthesis of oxytetracycline, a topic that has been debated in the literature. Loss of the cyclase protein has a profound influence on the length of polyketide chain assembled, implying that OtcD1 plays a greater role in the overall integrity of the quaternary structure of the polyketide complex than hitherto imagined.
( 2007 )
Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase.
PMID : 17137716 : DOI : 10.1016/j.bbagen.2006.10.011
We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the alpha beta alpha-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL=590 at 25 degrees C, pH=7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the beta-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.
( 1990 )
Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus.
PMID : 1700267 : DOI : 10.1007/bf00283033
The nucleotide sequence of a 1105 bp Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA(Gln) (CUG) genes, differing by 1 bp in the aminoacyl stem, and three identical tRNA(Glu) (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glu1-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3' terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3' terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.
( 2006 )
Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase.
PMID : 16597959 : DOI : 10.1128/AEM.72.4.2573-2580.2006 PMC : PMC1449064
Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.
( 2005 )
Update on acquired tetracycline resistance genes.
PMID : 15837373 : DOI : 10.1016/j.femsle.2005.02.034
This mini-review summarizes the changes in the field of bacterial acquired tetracycline resistance (tet) and oxytetracycline (otr) genes identified since the last major review in 2001. Thirty-eight acquired tetracycline resistant (Tc(r)) genes are known of which nine are new and include five genes coding for energy-dependent efflux proteins, two genes coding for ribosomal protection proteins, and two genes coding for tetracycline inactivating enzymes. The number of inactivating enzymes has increased from one to three, suggesting that work needs to be done to determine the role these enzymes play in bacterial resistance to tetracycline. In the same time period, 66 new genera have been identified which carry one or more of the previously described 29 Tc(r) genes. Included in the new genera is, for the first time, an obligate intracellular pathogen suggesting that this sheltered group of bacteria is capable of DNA exchange with non-obligate intracellular bacteria. The number of genera carrying ribosomal protection genes increased dramatically with the tet(M) gene now identified in 42 genera as compared with 24 and the tet(W) gene found in 17 new genera as compared to two genera in the last major review. New conjugative transposons, carrying different ribosomal protection tet genes, have been identified and an increase in the number of antibiotic resistance genes linked to tet genes has been found. Whether these new elements may help to spread the tet genes they carry to a wider bacterial host range is discussed.
( 2005 )
Ablation of the otcC gene encoding a post-polyketide hydroxylase from the oxytetracyline biosynthetic pathway in Streptomyces rimosus results in novel polyketides with altered chain length.
PMID : 16148009 : DOI : 10.1074/jbc.M503191200
Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC "minimal" polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex.
( 2004 )
Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry.
PMID : 15653427 : DOI : 10.1515/BC.2004.148
The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus . The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Deltam=0.11 Da and Deltam=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.
( 2004 )
Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine.
PMID : 15578758 : DOI : 10.1002/jms.750
A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H](2+) ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme.
Leš?i? Ašler I,
( 2010 )
Probing enzyme promiscuity of SGNH hydrolases.
PMID : 20931591 : DOI : 10.1002/cbic.201000398
Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.
( 2009 )
Identification of OxyE as an ancillary oxygenase during tetracycline biosynthesis.
PMID : 19472250 : DOI : 10.1002/cbic.200900122
The double hydroxylation of 6-pretetramid to 4-keto-anhydrotetracycline is a key tailoring reaction during the biosynthesis of the broad-spectrum antibiotic tetracyclines. It has been shown previously by heterologous reconstitution that OxyL is a dioxygenase and is the only enzyme required to catalyze the insertion of oxygen atoms at the C-12a and C-4 positions. We report here that OxyE, a flavin adenine dinucleotide (FAD)-dependent hydroxylase homologue, is an ancillary mono-oxygenase for OxyL during oxytetracycline biosynthesis in Streptomyces rimosus. By using both gene disruption and heterologous reconstitution approaches, we demonstrated that OxyE plays a nonessential, but important role in oxytetracycline biosynthesis by serving as a more efficient C-4 hydroxylase. In addition, we demonstrated that partially oxidized biosynthetic intermediates can undergo various glycosylation modifications in S. rimosus. Our results indicate that the synergistic actions of OxyE and OxyL in the double hydroxylation step prevent accumulation of shunt products during oxytetracycline biosynthesis in S. rimosus.
( 2008 )
Deciphering deazapurine biosynthesis: pathway for pyrrolopyrimidine nucleosides toyocamycin and sangivamycin.
PMID : 18721750 : DOI : 10.1016/j.chembiol.2008.07.012 PMC : PMC2603307
Pyrrolopyrimidine nucleosides analogs, collectively referred to as deazapurines, are an important class of structurally diverse compounds found in a wide variety of biological niches. In this report, a cluster of genes from Streptomyces rimosus (ATCC 14673) involved in production of the deazapurine antibiotics sangivamycin and toyocamycin was identified. The cluster includes toyocamycin nitrile hydratase, an enzyme that catalyzes the conversion of toyocamycin to sangivamycin. In addition to this rare nitrile hydratase, the cluster encodes a GTP cyclohydrolase I, linking the biosynthesis of deazapurines to folate biosynthesis, and a set of purine salvage/biosynthesis genes, which presumably convert the guanine moiety from GTP to the adenine-like deazapurine base found in toyocamycin and sangivamycin. The gene cluster presented here could potentially serve as a model to allow identification of deazapurine biosynthetic pathways in other bacterial species.
( 2008 )
Identifying the minimal enzymes required for anhydrotetracycline biosynthesis.
PMID : 18422316 : DOI : 10.1021/ja800951e
The cyclohexenone ring A of tetracyclines exhibits unique structural features not observed among other aromatic polyketides. These substitutions include the C2 primary amide, C4 dimethylamine, and the C12a tertiary alcohol. Here we report the identification and reconstitution of the minimum set of enzymes required for the biosynthesis of anhydrotetracycline (ATC, 5), the first intermediate in the tetracycline biosynthetic pathway that contains the fully functionalized ring A. Using a combination of in vivo and in vitro approaches, we confirmed OxyL, OxyQ, and OxyT to be the only enzymes required to convert 6-methylpretetramid 1 into 5. OxyL is a NADPH-dependent dioxygenase that introduces two oxygen atoms into 1 to yield the unstable intermediate 4-keto-ATC 2. The aminotransferase OxyQ catalyzes the reductive amination of C4-keto of 2, yielding 4-amino-ATC 3. Furthermore, the N, N-dimethyltransferase OxyT catalyzes the formation of 5 from 3 in a (S)-adenosylmethionine (SAM)-dependent manner. Finally, a "non-natural" anhydrotetracycline derivative was generated, demonstrating that our heterologous host/vector pair can be a useful platform toward the engineered biosynthesis of tetracycline analogues.
( 1991 )
Characterization of an oxytetracycline-resistance gene, otrA, of Streptomyces rimosus.
PMID : 1809836 : DOI : 10.1111/j.1365-2958.1991.tb01852.x
The sequence of a 2657 bp DNA fragment containing the coding and regulatory regions of the oxytetracycline (OTC)-resistance gene, otrA, from the OTC producer Streptomyces rimosus was determined. The predicted amino acid sequence of OtrA had extensive identity with tetracycline-resistance genes from other bacteria which mediate resistance via non-covalent ribosomal modification. The N-terminal domain had extremely high identity with the GTP-binding sites of elongation factors, such as EF-G and EF-Tu, suggesting that binding and hydrolysis of GTP is important to the function of the protein. Significant identity with EF-G was present throughout the polypeptide. Transcriptional activity upstream of the otrA coding region was investigated. An Escherichia coli-type promoter, otrAp1, was identified. Transcriptional readthrough of otrA from the upstream gene (otcZ) was also detected in S. rimosus cultures. A divergent promoter activity was identified with subclones of the OtrA fragment in promoter probe vectors analysed in Streptomyces lividans. However, this activity was not identified in a subclone containing more than half of the otrA coding sequence in S. lividans or at all in S. rimosus, indicating that OtrA negatively regulates the expression of the divergent transcript. The data are consistent with regulation of antibiotic production by OtrA to prevent 'suicide'.
( 2016 )
Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.
PMID : 27338640 : DOI : 10.1016/j.bbrc.2016.06.097
Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 ?. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding.
( 2012 )
[Cloning and expression of the redox-sensing transcriptional repressor Rex and in vitro DNA-binding assay of the Rex and rex operator in Streptomyces rimosus M4018].
PMID : 22489458 :
The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018. We cloned the rex of S. rimosus M4018 (Sr-rex) based on its homologoussequence in Streptomyces coelicolor A3(2) and its upstream rex operator (ROP) fragment using PCR and genome walking. An electrophoretic mobility shift assay (EMSA) was applied to analyze the regulation of rex to ROP in vitro. Sr-rex is 846 bp in length and has a 84% identity with the one in S. coelicolor A3(2) in amino acid sequence. It was deposited in Genbank under the accession number GQ849479. The expressed Sr-Rex by E. coli was mainly composed of alpha-helixes and beta-sheets, which was in compliance with the prediction. An Electrophoretic Mobility Shift Assay (EMSA) confirmed the specific binding activity of Sr-Rex with ROP. Meanwhile, we synthesized a 22 bp DNA fragment (ROP1) based on the minimal binding site of ROP. The maximal binding ratio of this fragment to Sr-Rex was 5:1 (molar). NADH negatively affected the binding activity, however, NAD+ had no impact on it. In S. rimosus M4018, the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD (H).
( 2012 )
Ribosomal and protein coding gene based multigene phylogeny on the family Streptomycetaceae.
PMID : 22154623 : DOI : 10.1016/j.syapm.2011.08.007
The phylogenetic relationship among the three genera of the family Streptomycetaceae was examined using the small and large subunit ribosomal RNA genes, and the gyrB, rpoB, trpB, atpD and recA genes. The total stretches of the analyzed ribosomal genes were 4.2kb, and those of five protein coding genes were 4.5 kb. The resultant phylogenetic trees confirmed that each genus formed an independent clade in the majority of cases. The G+C contents of rRNA genes were 56.9-58.9 mol%, and those of protein coding genes were 65.4-72.4 mol%, the latter being closer to those of the genomic DNAs. The average nucleotide sequence identity between the organisms were 94.1-96.4% for rRNA genes and 85.7-90.6% for protein coding genes, thus indicating that protein coding genes can give higher resolution than rRNA genes. In addition, the protein coding gene trees were more stable than the rRNA gene trees, supported by higher bootstrap values and other treeing algorithms. Moreover, the genome data of six Streptomyces species indicated that many protein coding genes exhibited higher correlations with genome relatedness. The combined gene sequences were also shown to give a better resolution with higher stability than any single genes, though not necessarily more correlated with genome relatedness. It is evident from this study that the rRNA gene based phylogeny can be misleading, and also that protein coding genes have a number of advantages over the rRNA genes as the phylogenetic markers including a high correlation with the genome relatedness.
( 1996 )
Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa.
PMID : 8760910 : DOI : 10.1099/13500872-142-8-1973
The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.
( 1994 )
Sequences of the oxytetracycline polyketide synthase-encoding otc genes from Streptomyces rimosus.
PMID : 8163168 : DOI : 10.1016/0378-1119(94)90144-9
The complete nucleotide sequences of the Streptomyces rimosus oxytetracycline (oxyTc) polyketide synthase (PKS)-encoding genes (otcY) has been determined, revealing three open reading frames. The deduced amino-acid sequences correspond to the presumed heterodimeric beta-ketoacyl synthase and acyl carrier protein found in other type-II (multicomponent) PKS systems that specify construction of acetate-derived polyketide antibiotics.
( 1988 )
Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus.
PMID : 3053973 : DOI : 10.1099/00221287-134-3-585
Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.
( 1989 )
Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.
PMID : 2914874 : DOI : 10.1128/jb.171.2.887-895.1989 PMC : PMC209679
The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.
( 1989 )
Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene from Streptomyces rimosus forma paromomycinus.
PMID : 2914849 : DOI : 10.1128/jb.171.1.321-328.1989 PMC : PMC209590
A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. The -10 and -35 regions of this fragment showed limited sequence resemblance to other Streptomyces promoters. The primary structure of the AAC(3)-VII enzyme showed strong homology with those of the AAC(3)-III and AAC(3)-IV enzymes encoded by plasmids in gram-negative bacterial genera. Upstream of the aacC7 gene was an open reading frame of 357 nucleotides which did not appear to be involved in controlling the expression of the aacC7 gene.
( 1998 )
Revised sequence of OtrB (tet347) tetracycline efflux protein from Streptomyces rimosus.
PMID : 9867793 : PMC : PMC105996
( 1997 )
The recA gene from Streptomyces rimosus R6: sequence and expression in Escherichia coli.
PMID : 9765818 : DOI : 10.1016/S0923-2508(97)83870-3
The recA gene from Streptomyces rimusus encodes a 376-amino acids polypeptide (M(r) 39,702) that is one of the largest bacterial RecA proteins observed. Detailed analyses of the Streptomyces RecA proteins showed that all possess an additional and unique C-terminal, rich in lysines and alanines, which can form an additional terminal alpha helix. Expression of the S. rimosus RecA protein in Escherichia coli FR333 (delta recA306) was demonstrated using antibodies raised against E. coli RecA protein; expression was possible only from the S. rimosus promoter. A Streptomyces-E. coli-like promoter sequence (TTGACA-18bp-TCTTAT) was found in the A+ T-rich region 135-165 base pairs upstream from the initiation codon and was related to Bacillus subtilis DNA damage-inducible promoters.