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PubMed1998

Construction of Escherichia coli-Streptomyces shuttle expression vectors for gene expression in Streptomyces.

pIJ4123 and pIJ6021 are high-copy-number vectors for gene expression in Streptomyces. They contain a strong, thiostrepton-inducible promoter, PtipA. Two E. coli-Streptomyces shuttle vectors, pHZ1271 and pHZ1272, were constructed by inserting a replicon and bla gene from E. coli into pIJ4123 and pIJ6021, respectively. Both vectors were structurally stable either in E. coli or in Streptomyces lividans. To demonstrate the utility of pHZ1272, the hemoglobin gene of Vitreoscillia (vhb) was cloned into pHZ1272 and expressed in Streptomyces lividans. The expression product (VHb) was detected by Western blotting and carbon monoxide binding activity analysis.

Amino Acid Sequence↗
PubMed1994

Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification.

The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.

Amino Acid Sequence↗
PubMed2004

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66.

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.

Base Sequence↗
PubMed1981

Excision of chromosomal DNA sequences from Streptomyces coelicolor forms a novel family of plasmids detectable in Streptomyces lividans.

When strains of Streptomyces coelicolor A3(2) lacking the previously identified autonomous plasmids SCP1 and SCP2 are crossed with Streptomyces lividans 66, some of the S. lividans progeny are able to elicit zones of growth inhibition (lethal zygosis), previously associated with the transfer of conjugative Streptomyces plasmids, when grown in contact with S. lividans 66. Some such progeny yield covalently closed circular (CCC) plasmid DNA, the size and restriction endonuclease cleavage pattern of which is constant for a particular isolate, but varies among isolates. These plasmids, which have been named SLP1.1, SLP1.2, etc., all confer resistance to lethal zygosis elicited by the others. Genetic and molecular characterization of the plasmids reveals that they are derived from the strA region of the chromosome of S. coelicolor. It is proposed that, before or during mating with S. lividans, the SLP1 sequences are excised from the chromosome, bringing varying regions of the surrounding chromosome with them, and can circularize to yield the SLP1 family of plasmids. Autonomous SLP1 plasmids can also be generate by cleaving total DNA of S. coelicolor with certain restriction enzymes, ligating it, and transforming the DNA into S. lividans. The autonomous SLP1 plasmids exist within S. lividans in a few copies per chromosome, and act as fertility factors. They provide suitable vectors for DNA cloning since the segments of chromosomal DNA carried by the larger members of the family are dispensable.

Conjugation, Genetic↗
PubMed1982

Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans.

Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.

Bacteriophages↗
PubMed1996

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces.

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.

Amino Acid Sequence↗
PubMed1985

New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA.

The thiostrepton resistance gene (tsr) of Streptomyces azureus, and a synthetic oligonucleotide adapter sequence, were introduced into the DNA of attP-site-deleted phage phi C31-based cloning vectors. The DNA of two of the new derivatives, KC515 and KC516, contains single sites for the enzymes BamHI, BglII, PstI, PvuII, SstI (two sites close together) and XhoI, available for the insertion of DNA of up to 4 kb. The two vectors also contain a cloned, promoterless viomycin phosphotransferase gene (vph) from Streptomyces vinaceus. When an internal segment of the Streptomyces coelicolor glycerol (gyl) operon was inserted at the appropriate position and in the correct orientation next to vph, it could bring about in vivo recombination leading to fusion of vph of the chromosomally located gyl operon, resulting in glycerol-regulated expression of viomycin resistance. Two other new phi C31 derivatives, KC505 and KC518, are PstI and BamHI replacement vectors, respectively, for 2-8-kb DNA fragments, and allow simple screening for the presence of inserted DNA.

Bacteriophages↗
PubMed2004

Biochemical characterization of the glucose kinase from Streptomyces coelicolor compared to Streptomyces peucetius var. caesius.

Glucose kinase (Glk) from Streptomyces coelicolor was purified, characterized biochemically and kinetically, and compared to Glk from Streptomyces peucetius var. caesius. In both Streptomyces, 96% of the enzyme activity was detected in the cytosolic fraction. The use of a Glk activity gel showed no isoforms of the enzyme in extracts from these microorganisms. The purified S. coelicolor Glk was stable in its tetrameric form, unlike the purified enzyme from S. peucetius var. caesius which easily dissociated into dimers. Tetramer dissociation was prevented by 100 mM d-glucose; however, this effect was not observed with other sugars. The optimum pH and the pI are practically identical for both enzymes. Maximum activity was found at a lower temperature for S. coelicolor Glk (33 compared to 42 degrees C) and its activity was apparently less stable at higher temperatures. Its activation energy was also 40% lower than that of S. peucetius var. caesius. The kinetic mechanism appears to follow a rapid equilibrium-ordered Bi-Bi sequential mechanism in both microbial enzymes, where Glk first binds glucose and then the MgATP(2-) complex, to form a ternary complex (enzyme d-glucose-MgATP(-2)). The Km values for D-glucose and MgATP(2-) were 1.4, 0.5 mM and 1.6, 0.8 mM for the S. coelicolor and S. peucetius var. caesius Glks, respectively. However Vmax of S. coelicolor Glk was higher. In conclusion, the S. coelicolor Glk showed a more stable tetrameric form with better affinity for its substrates and higher Vmax, suggesting greater catalytic efficiency.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2).

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.

Chromosomes, Fungal↗
PubMed1991

Actinorhodin production by Streptomyces coelicolor and growth of Streptomyces lividans are improved by the expression of a bacterial hemoglobin.

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Anthraquinones↗
PubMed1998

Streptomyces pneumonia in a patient with human immunodeficiency virus infection: case report and review of the literature on invasive streptomyces infections.

Streptomyces species are most widely known for their production of antimicrobial substances and, apart from mycetoma, have rarely been reported as a cause of infection. We describe a patient with early human immunodeficiency virus infection who presented with fever, cough, and nodular pulmonary infiltrates. Open lung biopsy revealed necrotic tissue and a sulfur granule; aerobic bacterial cultures yielded Streptomyces species. The patient was treated successfully with clarithromycin for 6 months. We review the clinical presentation and treatment of invasive streptomyces infections.

AIDS-Related Opportunistic Infections↗
PubMed1997

Streptomyces cellulolyticus sp. nov., a new cellulolytic member of the genus Streptomyces.

Actinomycete strain LXT (T = type strain), which decomposes cellulose, was identified as a member of the genus Streptomyces on the basis of morphological characteristics and the chemotype of the cell wall. The key characteristics of this organism are rectiflexibiles spore chains, a nonfragmenting vegetative mycelium, a warty spore surface, a type I cell wall, white to pink spore masses, and a lack of formation of soluble pigments (including melanin). These results indicate that strain LXT represents a distinct Streptomyces species, for which the name Streptomyces cellulolyticus is proposed. The type strain is strain LX, which has been deposited in the China General Microbiological Culture Collection Center as strain AS. 41332.

Biodegradation, Environmental↗
PubMed1990

Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae.

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

Amidohydrolases↗
PubMed2003

Kitasatospora putterlickiae sp. nov., isolated from rhizosphere soil, transfer of Streptomyces kifunensis to the genus Kitasatospora as Kitasatospora kifunensis comb. nov., and emended description of Streptomyces aureofaciens Duggar 1948.

A polyphasic study was undertaken to establish the taxonomic position of a rhizosphere isolate that had been assigned provisionally to the genus Kitasatospora. The organism, isolate F18-98(T), was found to have chemical and morphological properties that were consistent with its classification as a Kitasatospora strain. Direct 16S rDNA sequence data confirmed the taxonomic position of the strain, following the generation of phylogenetic trees by using three treeing algorithms. The organism formed a 16S rDNA subclade with Kitasatospora azatica and Streptomyces kifunensis, but was distinguished readily from the latter by using a combination of biochemical and physiological properties. Genotypic and phenotypic data show that strain F18-98(T) should be classified in the genus Kitasatospora as a novel species, for which the name Kitasatospora putterlickiae sp. nov. is proposed; the type strain has been deposited in culture collections as strain F18-98(T) (=DSM 44665(T)=NCIMB 13932(T)). It is also proposed that Streptomyces kifunensis should be transferred to the genus Kitasatospora as Kitasatospora kifunensis comb. nov. An emended description of Streptomyces aureofaciens Duggar 1948 is also given.

Actinomycetales↗
PubMed1994

Expression of an heterologous gene activating actinorhodin biosynthesis in Streptomyces lividans and Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin resulted in activation in the non-producer strain Streptomyces lividans, but not in the natural producer strain Streptomyces coelicolor, when transformed with an heterologous activator gene from Streptomyces fradiae. The gene encodes a 132 nucleotide-long transcript, responsible for the actinorhodin production phenotype, and thought to act as a putative antisense RNA, which has been detected in the transformed S. lividans cultures by reverse transcription followed by cyclic amplification.

Anthraquinones↗
PubMed2003

Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts.

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

Bacterial Proteins↗
PubMed1983

Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.

4-Butyrolactone↗
PubMed1992

Characterization of the Streptomyces clavuligerus argC gene encoding N-acetylglutamyl-phosphate reductase: expression in Streptomyces lividans and effect on clavulanic acid production.

The argC gene of Streptomyces clavuligerus encoding N-acetylglutamyl-phosphate reductase (AGPR) has been cloned by complementation of argC mutants Streptomyces lividans 1674 and Escherichia coli XC33. The gene is contained in an open reading frame of 1,023 nucleotides which encodes a protein of 340 amino acids with a deduced molecular mass of 35,224 Da. The argC gene is linked to argE, as shown by complementation of argE mutants of E. coli. Expression of argC from cloned DNA fragments carrying the gene leads to high levels of AGPR in wild-type S. lividans and in the argC mutant S. lividans 1674. Formation of AGPR is repressed by addition of arginine to the culture medium. The protein encoded by the argC gene is very similar to the AGPRs of Streptomyces coelicolor, Bacillus subtilis, and E. coli and, to a lesser degree, to the homologous enzymes of Saccharomyces cerevisiae and Anabaena spp. A conserved PGCYPT domain present in all the AGPR sequences suggests that this may be the active center of the protein. Transformation of S. clavuligerus 328, an argC auxotroph deficient in clavulanic acid biosynthesis, with plasmid pULML30, carrying the cloned argC gene, restored both prototrophy and antibiotic production.

Aldehyde Oxidoreductases↗